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UNIVERSITY OF VETERINARY AND PHARMACEUTICAL SCIENCES BRNO FACULTY OF VETERINARY HYGIENE AND ECOLOGY Department of Biology and Wildlife Diseases HANDBOOK FOR BIOLOGY AND GENETICS PRACTICAL COURSES Eva Bártová Eva Roubalová Brno 2009 INTRODUCTION This handbook is addressed to the students of the English Master's degree study programme, at the University of Veterinary and Pharmaceutical Sciences in Brno. The book contains protocols for practical courses of “Biology and genetics I and II” that are focused on general biology and on the basis of genetics. We hope that this book will be helpful for you and that it will give you some feeling of the need for scientific knowledge, and how it can be implemented into practice. Protocols are divided into chapters (see “Content”) corresponding to particular lessons. Each chapter contains theoretical introduction, summary of important information, terms and definitions to the respective subject matter followed by individual tasks related to the respective chapter. This introduction is not an exhaustive review of the particular topic. To successfully pass each lesson, you are expected to comprehend the theoretical bases of the chapter by the study of both materials concerning corresponding lecture and additional materials before every lesson (for recommendations of suitable textbooks and study materials see the list at the end of this book). A great deal of the tasks includes microscopic examination. Therefore it is necessary to know how to work with the microscope, with its setting and with various microscopic techniques. You will also master the techniques of microscopic samples preparation (both native and permanent) and you will learn how to design, carry out and evaluate simple biological experiments. In one of the lessons you will learn the most important methods of molecular biology and you will see practical applications of these methods. You will also get the basic information about animal experiments, animal welfare and other related issues, including excursions to user facilities, where experimental animals are kept. Genetic section of the practicals will be based on exemplary genetic tasks solving, but you will also experience e.g. designing and interpretation of Drosophila melanogaster crossing and much more. Your success as a student in this course will require regular attendance, careful note taking (for style of protocol see chapter 1) and the mastery of each particular topic through careful study. You will spend 90 minutes every week in the laboratory practicing your acquired knowledge, therefore you have to study the relevant topics in advance. We strongly recommend you attending the lectures regularly, even if your presence on the lectures is not compulsory. The information provided there gives you the basis for your studies and the personal contact with your teacher gives you the opportunity to discuss the questionable topics, which will help you to make sense of the acquired information more easily. If questions or problems arise, do not hesitate to contact your teacher. We are always willing to talk with you and, if possible, assist you with your concerns. We wish you a successful and exciting first year at our university and all the best in all your academic pursuits. Authors 2 CONTENTS 1. Making records from the lessons ........................................................................................5 2. Methods used for obtaining information in biological sciences ...........................................6 3. Magnifying devices (magnifying glass, light microscope, electron microscope) .................7 3.1. Magnifying glass .........................................................................................................7 3.2. Light (optical) microscope ...........................................................................................8 3.2.1. History of light microscopes ..................................................................................8 3.2.2. Parts of a light microscope ....................................................................................8 3.2.3. Types of light microscopes .................................................................................. 14 3.3. Electron microscopes ................................................................................................. 17 3.3.1. Evolution of electron microscopes .......................................................................17 3.3.2. Types of electron microscopes............................................................................. 17 3.4. Microphotography .....................................................................................................19 4. Microscopic technique .....................................................................................................20 4.1. Dry objectives, centring the objects, iris diaphragm function .....................................20 4.2. Optical planes, measuring the size and thickness of microscopic objects .................... 23 4.3. Permanent preparations.............................................................................................. 25 4.4. Native preparation, phase contrast ............................................................................. 28 5. Prokaryotes and immersion microscopy ........................................................................... 31 5.1. Prokaryotes................................................................................................................ 31 5.1.1. Domain Bacteria ................................................................................................. 31 5.1.2. Domain Archaea ................................................................................................. 33 5.2. Observation using immersion objective .....................................................................34 6. Chemical composition of bioplasm .................................................................................. 37 6.1. Elements.................................................................................................................... 37 6.2. Chemical compounds ................................................................................................ 37 7. Non-cellular life ............................................................................................................... 42 8. Eukaryotes ....................................................................................................................... 46 8.1. Plant cell.................................................................................................................... 49 8.2. Animal and protozoan cells........................................................................................ 50 9. Research methods in biology............................................................................................ 53 9.1. Cell and tissue cultures .............................................................................................. 53 9.2. Molecular biology techniques .................................................................................... 56 9.3. Care of laboratory animals and animal experiments ................................................... 64 10. Transport of substances, osmosis .................................................................................... 68 11. Cell growth and reproduction ......................................................................................... 71 11.1. Mitosis in plant cell ................................................................................................. 74 11.2. Mitosis in animal cell ............................................................................................... 75 12. Movement and irritation ................................................................................................. 77 12.1. Movement ............................................................................................................... 77 12.2. Irritation .................................................................................................................. 80 13. Reproduction and development ...................................................................................... 82 13.1. Development ........................................................................................................... 84 13.2. Meiosis .................................................................................................................... 86 13.2.1. Meiosis in humans ............................................................................................. 87 14. Influence of surroundings onto the bioplasm ..................................................................92 14.1. Physical stress .......................................................................................................... 92 14.2. Chemical stress ........................................................................................................94 15. Genetics ......................................................................................................................... 96 15.1. Cytogenetics - study of chromosomes, karyotypes ................................................... 97 3 15.2. Model organism - Drosophila melanogaster .......................................................... 102 15.3. Monohybridism ..................................................................................................... 103 15.4. Dihybridism, polyhybridism and branching method ............................................... 106 15.5. Polymorphic genes................................................................................................. 108 15.6. Gene interactions ................................................................................................... 109 15.7. Inheritance and sex ................................................................................................ 112 15.8. Genetic linkage ...................................................................................................... 114 15.9. Population genetics ................................................................................................ 116 15.10. Quantitative genetics ............................................................................................ 118 16. Recommended literature............................................................................................... 120 4 1. Making records from the lessons You have to elaborate a protocol from each lesson onto an undersigned A4 sized paper or into notebook. Each protocol has to contain the date and topic of the lesson, followed by the individual tasks inscribed with number and title and containing drawings of observed objects. The drawings have to be done by a pencil (colored parts can be drawn by a crayons), they have to be large enough (two pictures on one page at the most) and have to contain a description (also by a pencil). The magnification used for the observation has to be pointed out alongside each drawing. In case you prepare the sample yourself, or if you perform an experiment, the protocol has to contain a description of the procedure. The outcome of an observation or the findings resulting from an experiment has to be summarized in “Conclusion”. The protocols have to be at university level including handwriting and overall appearance! Submission of complete and accurate protocols at the end of each semester (along with regular attendance and with successful passing of specified tests) is one of the requirements for granting your credit. It is necessary to elaborate the protocol(s) even in case of your absence in the particular lesson. PROTOCOL: Date: Topic of the practical work: TASK 1: (the title of the task) Procedure: Drawing: (sufficiently large, with a pencil and also a crayon, description) Magnification (10/4, 10/10, 10/40 and 10/100 or 40x, 100x, 400x and 1000x): Conclusion: (result and evaluation of observation or experiment) TASK 2: ............. 5 2. Methods used for obtaining information in biological sciences Each scientific branch, including biology, makes prompt efforts to obtain new information, that can supplement or extend current level of knowledge, or that can be instrumental to the verification of scientific hypotheses. In general biology, the main method for obtaining new data is the observation. For observation we can use all our senses (above all the sight), efficiency of which can be multiplied using various instruments (e.g. magnifying glass or a microscope). In biological and medical sciences, more complicated methods, requiring special knowledge and techniques, can be used for obtaining information. These include clinical, biochemical, serological, haematological, immunological, microbiological (bacteriological, virological, parasitological), cytogenetic, histologic or pathoanatomic examinations. It is also possible to employ various procedures involving animals, bacterial or eukaryotic cell cultures, or distinct methods of molecular biology. Additional information can be acquired from medical records, statistics, interviews, and questionnaires, from scientific and professional journals and from various databases available on the internet. The most important biochemistry, virology, haematology, bacteriology, cytology, cytogenetic and molecular biology methods will be detailed in separate chapters. Another method used for obtaining data is an experiment, which is a research method used for the determination of consequences induced in an experimental object by alteration of one of the factors that influence the object. The experiment is performed in the context of solving a particular problem or question, to retain or disprove a hypothesis or research concerning phenomena. Well defined and stable conditions have to be kept during the whole experiment to enable its reproducibility. Usually several replicate samples (duplicates, triplicates…) and both a positive and a negative control are included in an experiment. The results from replicate samples are often averaged, or if one of the replicates is obviously inconsistent with the others, it can be discarded as being the result of an experimental error. A positive control is a procedure that is very similar to the actual experimental test but which is known from a previous experience to give a positive result. The positive control confirms that the basic conditions of the experiment were able to produce a positive result, even if none of the actual experimental samples produce a positive result. The negative control demonstrates the base-line result obtained when a test does not produce a measurable positive result. The value of the negative control is often considered as a "background" value and can be subtracted from the test samples results. Various statistic methods are used to evaluate the results of an experiment. Since one of the main goals of Practice in Biology and Genetics is to learn to observe and classify some nature's phenomena at the microscopic level, the first chapters are applied to the microscopic technique. 6 3. Magnifying devices (magnifying glass, light microscope, electron microscope) The studies of microscopic structures and their functions require the use of various magnifying devices enabling observation of details that are normally below the resolution limits of the human eye (0.2 mm). Optical magnifying devices e.g. magnifying glass, light microscope can enlarge the image of an object 2-2000× depending on the optical system used and on the number and type of lenses employed. The resolution limit is up to 0.2 μm, enabling observation of the eukaryotic and prokaryotic cell. LENSES are simple optical devices with axial symmetry which transmit and refract light, concentrating or diverging the light beams. Lenses are usually made of glass or other suitable transparent material, such as plastic, fluorite (CaF2), synthetic resin, etc. Lenses are usually spherical, which means that their two surfaces are parts of the surfaces of spheres. Each surface can be convex (bulging outwards from the lens), concave (depressed into the lens), or planar (flat). If the lens is biconvex or plano-convex, a collimated or parallel beam of light passing through the lens in parallel with its axis and passing through the lens will be converged (focused) to a spot on the axis, at a certain distance behind the lens (known as the focal length); the lens is thus called a positive or converging (connecting) lens. Such lenses enlarge the image of the observed object. If the lens is biconcave or plano-concave, a collimated beam of light passing through the lens is diverged (spread). In this case, the lens is called a negative or diverging (dispersing) lens. Negative lenses make the image of the observed object smaller. Using an electron microscope we can achieve magnification of about 2 000 000× with the resolution limit up to 2-20 nm, enabling observation of e.g. viruses, that have the average size of about 50 nm. Electron microscopes use electrons to illuminate a specimen and electrostatic and electromagnetic lenses to focus the electron beam, thus creating an enlarged image of the observed object. Electron microscopes have much greater resolving power than light microscopes due to the wavelength of an electron, which is much smaller than that of a photon. The main disadvantages of the electron microscopes are that they are expensive to build and maintain and that they are very sensitive to external influence e.g. vibrations or magnetic fields and that the sample preparation is quite complicated and samples have to be viewed in a vacuum which disables observation of living objects. 3.1. Magnifying glass The magnifying glass is the simplest optical device giving only a small magnification, thus serving for observation of e.g. flower parts, small insects or plankton. Magnifying glasses consist of one or more positive (converging) lenses, magnifying 2-30×. The image of the observed object is acquired using a magnifying glass that is direct (not inverted) and magnified. 7 3.2. Light (optical) microscope 3.2.1. History of light microscopes On September 17, 1683, Dutch amateur microscope inventor Antonie van Leeuwenhoek (1632-1723) wrote to the Royal Society in London about his observations of the samples he prepared from the teeth plaque. Using his simple microscope he found "…an unbelievably great company of living animalcules, swimming more nimbly than any I had ever seen up to this time. The biggest sort bent their body into curves in going forwards. Moreover, the other animalcules were in such enormous numbers, that all the water seemed to be alive". The optical part of this microscope contained only one lens, the sample was placed on a tip in front of the lens and the microscope was held in a hand, close to the eye. This type of microscope magnified up to 270×, reached the resolution of up to 1.35 μm. But Leeuwenhoek was not the first microscope inventor. Around 1595 another Dutch, Zacharias Janssen invented a microscope formed by two lenses placed on the opposite ends of a movable tube. Nevertheless, this microscope magnified only 9× and showed serious optical defects. Microscopes invented by Englishman Robert Hook (1635-1703) reached higher magnifications, however the image quality was low. Optical defects of microscopes were corrected as late as in the 19th century, when Carl Zeiss Company began to manufacture microscopes using glass with improved features. Early microscopes were monocular, with an external light source (daylight, lamp) and a mirror. The principle of the optical microscope has not changed since the 19th century, when it had already reached its limits given by physical laws: magnification up to 2000× and resolution up to 0.2 µm. The light microscope is an optical device consisting usually of two systems of lenses: the eyepiece and objective. The light microscope can be monocular (for observation with one eye) or binocular (for two eyes). The latter one is used more often nowadays. In our lessons we will use transmission microscopy which means that the light passes through thin (transparent) objects. Therefore we usually observe small objects in thin layers of medium, thin sections of tissues, smears, compressed objects, etc. 3.2.2. Parts of a light microscope 1. Optical part – eyepiece (ocular) and objective 2. Illumination part – light source, condenser, condenser iris diaphragm, filters and mirror 3. Mechanical part (frame) – base, arm, tube, revolving nosepiece, stage with specimen holder, coarse and fine focus adjustment knobs OPTICAL PART The optical part of microscope includes a system of lenses (or by single lens) that form the eyepieces (lens close to eye) and objectives (the lens close to the object). The objective produces an enlarged, reversed and real image of the observed object which is then seen through the eyepiece and an even more enlarged, reversed, but virtual image is obtained. OBJECTIVES are optic systems composed of several lenses placed in a metal cover. Characteristic features of objectives: Focal length (f) indicates the distance between the lens and the focus. It ranges usually from 1.5 mm (the most magnifying objectives) to 20 mm. Magnification (M) = 250/f, where 250 is conventional working distance of the human eye in mm. It is the ratio between the apparent size of an object and its true size. It is 8 a dimensionless number. The magnification of an objective depends upon its focal length. The shorter is the focal length, the bigger magnification (and the opposite). Free working distance is the distance between the front lens of the objective and the observed object. The greater the magnification of the objective, the shorter the free working distance. Size of the field of view (visual field) decreases with increasing magnification. The greater the magnification, the smaller the field of view. Numerical aperture (NA) = n.sinα, where n is the refractive index of the substance between the objective and the sample and α is the entrance angle of the objective (see Fig. 1). NA is also related to the resolution (the higher NA, the better resolution). NA is an important technical/optical characteristic of the objective and its value is recorded on each objective. Aperture is the ability of the objective to accept (catch) as many light beams as possible. Aperture literally means “the opening“. s´ α Dry objective Immersed objective r´ Immersion oil Cover glass Slide glass A B r s Fig. 1: A – (α) is half the maximal angle under which the objective lens collect light from the object (entrance angle). B – The effect of refractive index of the environment between the objective lens and the cover glass of the sample (comparison of dry and immersion objectives) on the numerical aperture of the objective. In case of dry objective, the transversal ray (r) refracts at the glass/air interface and thus the light does not enter the objective (r´). In case of an immersion objective, the analogical light ray (s) goes straight through slide and cover glass and through immersion oil (lens glass, specimen glass and immersion oil have very similar refractive indexes, thus light rays do not refract) and enters the objective (s´). Resolution (D) is the ability of the objective to distinguish two closely situated points as separate points. D = NA/λ, where NA is a numerical aperture and λ is wave length of used light. This formula can also be written in a different way, depending on how you define D. If you express D in length units (nm, µm), you must use reversed version: D = λ/NA. The resolution of the light microscope is generally limited by the wave length of visible light, which is about 0.2 µm. Lens speed (lens opening) describes the ability of a lens to retain the light rays. It is a qualitative concept related to the relative aperture diameter. A lens may be referred to as "fast" or "slow" depending on its maximum aperture compared to a lens of similar focal length. Lens speed is given by the minimum relative aperture. A lens with a larger maximum aperture is a fast lens because it delivers more light intensity (illuminance) to the focal plane. Lens speed depends on the numerical aperture and on the size of the cone angle (2xα). The amount of light that enters the objective is also dependent on the refraction index (n) of the substance as light rays go through. In case the light passes through slide and cover glass of the sample (n = 1.5) and subsequently through air (n = 1), the light rays refract and some of them miss the objective and are lost. Lens speed can be increased by homogenizing the optical features (refractive indices) of the substance as the light rays go through, e.g. by the use of immersion oil (see Fig. 1). Among the substances that are used for immersion microscopy are e.g. cedar tree oil (n = 1.516), 9 Canada balsam (n = 1.515 – 1.530) or synthetic immersion oils, that are more commonly used. Refraction index of water is n = 1.33. Penetration ability (called depth of field in photographic objectives) is the ability of an objective to render a sharp image of several optical planes of the object at the same time. It is inversely related to NA and thus high magnification objectives have small penetration ability and lower magnification means greater penetration ability. The more you open the iris diaphragm (wider opening = greater aperture), the greater the penetration ability. It is therefore necessary to start the search for inconspicuous objects (such as scattered transparent epithelial cells, or pollen grains) with a damped (dim) light and to add more light when shifting to a greater magnification. Aberration of objectives: Chromatic aberration (or defect) is the phenomenon of different color focusing at different distances from a lens. It causes that an image in white light tends to have colored edges. Spherical aberration (or defect) is a deviation resulting in an image imperfection that occurs due to the increased refraction of light rays when rays strike a lens near its edge, in comparison with those that strike nearer the centre. It makes focusing of lenses less than ideal due to their spherical shape. Types of objectives: 1. According to the substance used between the front lens of the objective and the observed object: a) Dry – objectives with smaller magnification (usually 2-60×), that have air (n = 1) between objective and sample. b) Immersion – more magnifying objectives (usually 90-100×), that are immersed into the medium (medium is between objective and sample). The medium must be removed from both the objective and the permanent sample after finishing the observation using a cloth soaked with ethanol or xylen. Immersion objectives usually have spring-loaded tips that protect them from damage and are marked with a black ring. 2. According to optical features (degree of correction of various aberrations of lenses): a) Achromatic objectives are designed to limit the effects of chromatic aberration. They are corrected to bring two wavelengths (typically yellow and green) into focus in the same plane. Achromatic objectives belong to the most commonly used objectives. b) Planachromatic objectives specified as "plan", will show visual field that is focused both in the centre and at its periphery. Planachromatic objectives have been corrected for both achromatic and spherical aberrations. These objectives belong to the best and the most expensive and are used for microphotography. c) Apochromatic objectives are corrected for both chromatic and spherical aberrations. The chromatic aberration is usually corrected for green, yellow, blue and red colors, while spherical aberration is corrected for two colors. They are used for color microphotography. d) Monochromatic objectives are designed for observation in monochromatic light (light of a single wavelength). They can be used e.g. for microscopy using ultraviolet (UV) radiation. 10 Color markings of objectives: Objectives are usually marked by color stripes. The microscopes in the biology classrooms are equipped by the objectives that are typed in bold: Magnification 1 2 20 4 10 black brown red yellow green Color 40 blue 50 60 100 light blue cobalt blue white Description of an objective contains (see Fig. 2): Type of an objective (e.g. “A” - meaning that this objective is apochromatic with corrected chromatic aberration, PL - objective for phase contrast microscopy) Magnification (e.g. 40 ) Numerical aperture (0.65) Observation tube length (160 mm) Recommended thickness of the cover glass in mm (0.17 mm) Producer Type of objective (A - achromatic) PL - objective for phase contrast microscopy Numerical aperture Magnification Recommended thickness of the cover glas in mm Observation tube length in mm Fig. 2: Description of an objective. EYEPIECE (OCULAR) is a cylinder containing two or more lenses to bring the image into focus for the eye. The eyepiece is inserted into the top end of the tube. Eyepieces are interchangeable and many different eyepieces can be inserted with different degrees of magnification. Conventional eyepieces magnify 10×. An eyepiece can be equipped with eyepiece micrometer that enables the measuring of observed objects. Eyepiece can also have dioptric adjustment mechanism that allows users who wear eye-glasses to adjust their diopters so they don't need glasses for microscopic observation. The tube also enables the interpupillary distance adjustment that allows to change the distance between the two eyepieces to fit it for user’s individual distance between the centres of his/her pupils. TOTAL MAGNIFICATION (M) = M objective x M eyepiece, usually given as a fraction, e. g. 10/40. This means that a specimen viewed by the 40 magnifying objective is actually enlarged 400 . The magnification of light microscopes used in practicals ranges from 40× to 1000×. (Note: Every drawing of a microscopic object must be completed with these data!) Ocular Objective Total magnification Magnification 10× 10× 10× 40× 100× 400× 10× 4× 40× 11 10× 100× 1000× ILLUMINATION PART Illumination part provides the light to illuminate the sample and to regulate and homogenize light rays. LIGHT SOURCE - both daylight and artificial light (electric bulb) can be used for observation. In most of the modern microscopes, the light source is incorporated in the base of the microscope. CONDENSER is a system of lenses that concentrates the light in the form of a cone on the object (specimen) so that the observed area is equally illuminated. Its distance from the object should be the same as the distance between the object and the objective. However, in some microscopes the condenser is in the fixed highest position. IRIS DIAPHRAGM is a mechanism with an opening (aperture) at its centre, that is used to close and open the hole the light passes through from the light source to the condenser. Iris diaphragm has an adjustable opening (like the iris of human eye), that is shaped in a nearround fashion by a number of movable blades that can change the diameter of the opening, thus regulating the light intensity. MIRROR is important to direct and concentrate the light into the condenser. Microscopes with light source incorporated into the base usually have flat metal mirror incorporated into the base. FILTERS modify the light from the light source to make it suitable for direct observation or for microphotography. Protective filters (yellow or orange) protect eyes from harmful UV radiation, the yellow-green filter is used in phase contrast microscopy to monochromatize the light, polarization filters are used to polarize the light, blue filter made of cobalt glass is used to absorb the yellow component of the artificial light. MECHANICAL PART Mechanical part forms the frame of the microscope and carry the optical and illumination parts. BASE forms the foot of the microscope and harbours the light source, mirror and filter holder. ARM holds the revolving nosepiece, the tube with the eyepieces, the movable stage with specimen holder, and a condenser that is located below the stage. TUBE is connected to revolving nosepiece (on its bottom) and it carries eyepiece(s) on its top. Tubes can be monocular, binocular or even trinocular (one output can be used for redirecting the light into the camera, CCD or other recording device). REVOLVING NOSEPIECE (TURRET) is a part of the microscope that holds two or more different objectives. It is located on the bottom of the tube and it can be rotated to easily change the resolving power. STAGE is a platform below the objective which supports the specimen being viewed. In the centre of the stage, there is a hole enabling the passage of light from illumination parts, through the sample and further into the optical system. Stage is equipped with the movable specimen holder that facilitates searching for desired objects. SPECIMEN HOLDER enables moving the sample in two different directions (left-right and back-forward). It is usually scaled, which allows localization of appropriate region of the sample. COARSE AND FINE FOCUS ADJUSTMENT KNOBS are mounted on both sides of the arm and control the focusing by moving the stage up and down. The larger knurled wheel is to adjust coarse focus, while the smaller knurled wheel controls fine and accurate focusing. The fine focus adjustment wheel is equipped with a graded scale (with divisions at 2.5 µm each), that enables the measuring of the thickness of the observed objects. 12 Eyepiece/ocular Diopter adjustment ring Tube Observation tube securing knob Arm Revolving nosepiece Objective Vertical and horizontal feed knob Specimen holder Stage Coarse focus adjustment knob Condenser iris diaphragm dial Condenser Window lens Base Fine focus adjustment knob Main switch Brightness adjustment knob Fig. 3: Parts of a light microscope. 13 3.2.3. Types of light microscopes 1. According to the number of eyepieces: a) Monocular microscopes b) Binocular microscopes 2. According to the light course: a) Transmitted light microscopes (including inverted microscopes) b) Incident light microscopes (reflected light microscopes) c) Dissecting microscopes 3. According to the light source/illumination type: a) Phase contrast microscopy b) Polarized light microscopy c) Differential interference contrast microscopy d) Dark field microscopy e) Fluorescent microscopy f) Confocal microscopy Monocular microscopes can be used for observation by one eye, with the possibility to use the other eye for drawing. Binocular microscopes enable observation by both eyes. The light collected by the objective is divided by reflecting prisms (and/or a beam splitter) located in the tube equally into both eyepieces. Transmitted light microscopy – the light from the light source is reflected by the mirror into the condenser, where it is condensed into small area. Subsequently it passes through the sample into the objective. Note: microscopes used in the biology classrooms are binocular light (optical) microscopes for observation in transmitted light. Inverted (inversion) microscope – optical system of inversion microscope is „upside-down“, i.e. the light source and condenser are located above the sample, while objectives are below the stage. The inversion microscope is mainly used for observation of living cells in the cell or tissue cultures at the bottom of cultivation vessels (bottles, flasks). Incident light microscopy (reflected light microscopy) – this technique is used for the observation of opaque (non-transparent) objects (e.g. minerals). Because light is unable to pass through these specimens, it must be directed onto the surface and eventually returned to the microscope objective by either specular or diffused reflection. Dissecting microscopes are configured to allow low magnification of larger objects (objects larger or thicker than the compound microscope can accommodate). Dissecting microscopes are binocular and allow seeing objects in three dimensions (3D) i.e. in stereo. Dissecting microscopes utilize both incident light (direct illumination) and transmitted light, enabling observation of opaque objects. Microscopes can be equipped with additional accessories for special microscopic techniques (e.g. phase contrast microscopy, dark field microscopy, UV or polarized light microscopy). Phase contrast microscopy differs from normal transmitted light microscopy in an illumination technique in which a small phase shift in the light passing through transparent specimens is converted into amplitude or contrast changes in the image. This method does not require staining to view the objects, thus enabling to study structures and processes in living cells (e.g. the cell cycle). Phase contrast microscopy proved to be such advancement in microscopy that Dutch physicist Frits Zernike, who discovered this technique, was awarded the Nobel Prize in physics in 1953. In optical microscopy, many objects (e.g. cell parts in protozoan, bacteria or sperm flagella) are essentially fully transparent unless stained (and 14 therefore killed). The difference in densities and composition within these objects, however, often give rise to changes in the phase of light passing through them. The use of phase contrast technique makes these structures visible even in native preparations. Equipment for phase-contrast microscopy consists of diaphragm (a phase ring) located in the phase objective and a correspondent diaphragm, which is located in the phase condenser. Phase contrast illumination can be established only through correctly centring the two diaphragms. A phase microscope (also called an auxiliary microscope) that temporarily replaces one of the oculars is used to centre the objective and condenser diaphragms. When aligned properly, light waves emitted from the illumination source arrive at the eye 1/2 wavelength out of phase. The phase contrast microscope uses the fact that the some of the light passing through a specimen is diffracted (and/or refracted) and due to this is shifted compared to the uninfluenced light. This phase shift is not visible to the human eye. However, the change in the phase can be increased to half a wavelength using the phase condenser and the phase objective, that are inserted into the optical path of the microscope, causing a difference in brightness. This makes the transparent object shine out in contrast to its surroundings. In the positive phase contrast optics, the phase condenser reduces the amplitude of all light rays travelling through the phase annulus by 70 to 90 % and advances the phase by yet another 90° (λ/4). The recombination of these waves with waves that were not shifted results in a significant amplitude change at all locations where there is interference due to 180° (λ/2) phase shifted waves. The net phase shift of 180° (λ/2) results directly from the 90° (λ/4) retardation of the wave due to the phase objective and the 90° (λ/4) phase advancement of the wave due to the phase condenser. π B A C Fig. 4: A – wave length of light ( ) and π = /2, B – quarter a wavelength phase shift (1/4 , i.e. +π/2), C – three quarters a wavelength phase shift (3/4 , i.e. -π/2). B A Fig. 5: Interference: A – light beams in the same phase (sum), B – light beams in reverse phase (difference). Polarized light microscopy (polarizing microscopy) can distinguish between isotropic and anisotropic materials. Isotropic materials which include gases or liquids, demonstrate the same optical properties in all directions. They have only one refractive index and no restriction on the vibration direction of light passing through them. Anisotropic materials which include 90 % of all solid substances in contrast have optical properties that vary with the orientation of incident light with the crystallographic axes. They demonstrate 15 a range of refractive indices depending both on the propagation direction of light through the substance and on the vibrational plane coordinates. More importantly, anisotropic materials act as beam splitters and divide light rays into two parts. Polarizing microscopy exploits the interference of the split light rays, as they are re-united along the same optical path to extract information about these materials. Differential interference contrast microscopy (DIC) or Nomarski Interference Contrast (NIC) is used to enhance the contrast in unstained, transparent samples. It works by separating a polarized light into two beams which take slightly different paths through the sample. Where the lengths of each optical path differ, the beams interfere when they are recombined. This gives the appearance of a 3D physical relief corresponding to the variation of optical density of the sample, emphasizing lines and edges. Image produced using DIC helps to visualize otherwise invisible features. It is similar to image obtained by phase contrast microscopy but without the bright diffraction halo. Dark field microscopy (dark ground microscopy) describes microscopy methods (in both light and electron microscopy), which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e. where there is no specimen to scatter the beam) is generally dark. This microscopy technique requires special illumination parts used to enhance the contrast in unstained samples. It works on the principle of illuminating the sample with light that will not be collected by the objective lens, so not to form part of the image. Light enters the microscope for illumination of the sample. In dark field microscopy, specially sized disc (the patch stop) blocks some light from the light source, leaving an outer ring of illumination. The condenser lens focuses the light towards the samples; after the light enters the sample, most of it is directly transmitted, while some is scattered from the sample. The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected due to a direct illumination block. Only the scattered light goes on to produce the image, while the directly transmitted light is omitted. Fluorescent microscopy is a light microscopy technique that is using the phenomena of fluorescence and phosphorescence instead of (or in addition to) reflection and absorption. In most cases, a component of interest in the specimen is specifically labelled with a fluorescent molecule called a fluorophore (or fluorochrome) e.g. GFP (green fluorescent protein) or fluorescein. The specimen is illuminated with light of a specific wavelength(s) that is absorbed by fluorophore, causing it to emit longer wavelengths of light of a different color than the absorbed light. The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Typical components of a fluorescence microscope are the light source (xenon arc lamp or mercuryvapor lamp), the excitation filter, the dichroic mirror (or dichromatic beam splitter), and the emission filter. Also, many biological molecules (e.g. chlorophyll, haemoglobin or some vitamins) are naturally fluorescent, thus emitting light of visible wavelengths after induction by UV light. Confocal microscopy is an optical technique used to increase image contrast and/or to reconstruct 3D image by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that are thicker than the focal plane. Confocal microscopes also use fluorescence phenomenon and they use laser (Light Amplification by Stimulated Emission of Radiation) as a light source. They use “point illumination” and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information. Only the light within the focal plane can be detected, so the image quality is much better than that of “wide-field” images. As only one point is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen. The thickness of the focal plane is defined mostly by the square of the numerical aperture of the objective lens, and also by the optical properties of the 16 specimen and the ambient index of refraction. This microscopic technique enables dynamic observations, such as live cell imaging. 3.3. Electron microscopes 3.3.1. Evolution of electron microscopes Some objects, e.g. viruses, are too small to be observed even by the most powerful microscopes equipped with the best glass lenses. This was discovered by a German physicist Ernst Abbe in the 19th century. The resolving power of optical microscopes is limited by the wavelength of visible light that ranges from 380 to 750 nm. Thus even the best light microscopes are limited to magnifications of approximately 2000 times. Electron microscopes can magnify objects up to two million times due to the wavelength of an electron that is much smaller than that of a light photon, enabling to visualize individual atoms. The first prototypes of electron microscopes (EM) were constructed in the thirties of the 20th century and exceeded the resolution possible with optical microscopes. Construction of EM was enabled by technological progress in general and interconnected several discoveries of various investigators. One of them was the discovery of the electron by English physicist and Nobel laureate Sir Joseph John Thomson in 1897. The next step leading to the use of electrons to visualize atoms, molecules and other very small objects was the finding published by the French physicist Louis de Broglie in 1925. He introduced the theory of electron waves that included wave - particle duality theory of matter, based on the work of Albert Einstein and Max Planck. He stated that any moving particle or object (including electrons) had an associated wave and thus created a new field in physics - wave mechanics, uniting the physics of light and matter. The electron, carrying a negative electrical charge, is attracted by everything that is charged positively, which can be used to impart it certain velocity that corresponds to particular wavelength. Moreover, the trajectory of moving electron can be influenced by a strong electromagnetic field in like manner the light is influenced during its passage through optical lenses. These features predestined electrons to be used as the new “light” suitable for “microcosms” investigation. Subsequently, two types of electron microscopes (transmission and scanning) were constructed, both of them using beams of accelerated electrons. The first Transmission Electron Microscope was constructed in 1931 by German scientists Max Knoll and Ernst Ruska. In 1986, Ernst Ruska was awarded a Nobel Prize in Physics for his fundamental work in electron optics, and for the design of the first electron microscope. Electron microscopes have advanced the resolving power of up to tenths of nanometres, i.e. the dimensions of atoms. 3.3.2. Types of electron microscopes TRANSMISSION ELECTRON MICROSCOPE (TEM) involves a beam of electrons emitted by an electron gun (usually wolfram cathode). Electrons are accelerated by an anode, focused by electrostatic and electromagnetic lenses and transmitted through a specimen that is partially transparent to electrons and partially scatters them out of the beam. When electrons come through the specimen, they carry information about the structure of the specimen (the "image") that is magnified (about 50×) by the objective lens system of the microscope and by other lenses placed below the objective. The electron image formed by electrons that passes through the specimen is then recorded and projected onto a viewing screen coated with a small amount of fluorescent material (e.g. phosphorus). The image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or by a CCD (charge-coupled device) camera. 17 The image detected by the CCD may be displayed on a monitor or computer. The Image acquired by TEM is black and white. The operating temperature of an electron gun of TEM is about 2500°C. At such a temperature, interactions of electrons with matter would be very strong. Thus gas particles must be absent in the area where electrons proceed. The required high vacuum is maintained by a vacuum system consisting of various pumps (pre-vacuum pump, diffusion pump, ion getter pump). High Resolution TEMs (HRTEMs) allow the production of images with sufficient resolution to show e.g. carbon atoms in diamond separated by only 89 picometers at magnifications of 50 million times. Preparation of samples for observation by TEM is quite sophisticated and includes various procedures, such as chemical fixation, dehydration (e.g. by freeze drying or by replacement of water with organic solvents, such as ethanol or acetone) that prevents water evaporation; embedding (in resin); sectioning by ultramicrotome with a glass or diamond knife that produces ultra thin (about 90 nm thick) slices of specimen semitransparent to electrons and staining (treatment of samples by contrasting agents, such as lead citrate, uranyl acetate or other compounds containing heavy metals) to scatter imaging electrons and thus giving contrast to different structures (e.g. cell organelles), since biological materials are often too transparent to electrons and thus insufficiently contrastive. Electron gun Eye Eyepiece Condenser Sample Objective Objective Projector Sample Condenser CCD camera (photographic film) Light source Fig. 6: Principle of light microscope. Fig. 7: Principle of transmission electron microscope. SCANNING ELECTRON MICROSCOPE (SEM) is designed for direct studying of the surfaces of objects. By scanning with an electron beam that has been generated by heating of a metallic filament and focused by electromagnetic lenses, an image is formed in similar way as in TV. The electron beam is rastered across the sample and secondary electrons are emitted from the surface of the specimen due to excitation by the primary electrons. Detectors collect these secondary (or backscattered) electrons, mapping the detected signals with a beam position, and convert this information to a signal, thus building up an image. The most important step in sample preparation for observation by SEM is the conductive coating. Since SEM use electrons to produce an image, electrically conductive samples are required. The most widely used procedure for SEM sample preparation is the use of gold atoms to cover the sample surface. Biological samples are placed in a small vacuum chamber, inside of which an electric field is used to remove electrons from argon gas atoms to make positively charged ions that are attracted to 18 a negatively charged piece of gold foil. The argon ions are knocking gold atoms from the surface of the foil. These gold atoms settle onto the surface of the sample, producing a gold coating. The SEM resolution is about an order of magnitude smaller than resolution of TEM, however, the advantages of SEM are the relatively easy sample preparation, the ability to image bulky samples and much greater depth of view (compared to both TEM and optical microscope), producing attractive images that represent the 3D structure even of rugged samples, such as insects. Both light and electron microscopes have their advantages and disadvantages: Light (optical) microscopes use (visible) light to visualize the image. Light rays pass through the optical system of the microscope, usually formed by the glass lenses. The resolution (resolving power) of light microscope is up to 0.2 µm, with magnification up to 2000×. The advantage of light microscope is that it enables observation of native preparations without complicated sample preparation. Images acquired by optical microscope can be colored. Electron microscopes use electrons to visualize the image. Electron beams are controlled and focused by electrostatic and electromagnetic lenses. The average resolution of an electron microscope is about 2-20 nm, with the magnification approx. 2 000 000×. Among the disadvantages of the use of electron microscopes, we can mention their high purchase price, complicated and time-consuming sample preparation and the impossibility to observe living objects. Images acquired by EM are black and white. Favourable is the possibility to obtain 3D image by SEM. Electron gun Scanning generator Condenser Deflector Objective Electron s Screen Detector Sample orek Fig. 8: Principle of scanning electron microscope. 3.4. Microphotography Microphotography (photographic record of a microscopic image) can be divided into: a) Microphotography utilizing classical photographic materials processed in chemical way is employed mainly by users who want to obtain large format photographs. b) Digital microphotography processes digital record of an image. It is preferred by users who want to edit, analyze or archive their images using computer. 19 4. Microscopic technique 4.1. Dry objectives, centring the objects, iris diaphragm function EQUIPMENT FOR MICROSCOPIC OBSERVATION Laboratory glassware: slides (76 × 26 × 1-1.2 mm with or without cut edges), cover glasses or cover slips (square-shaped or rectangular, 18 × 18, 22 × 22, 30 × 40 mm), beakers, test tubes, Petri dishes, wash bottles, graduated cylinders, funnels. Instruments: microscope, water bath, magnifying glass, burner, scissors, scalpel, tweezers, inoculation loop, teasing needle and droppers. Chemicals: dyes and stains, acids, alkalies and their salts, immersion oil, glycerol. MICROSCOPIC OBSERVATION STEP BY STEP: 1. Remove the dust cover from your microscope. 2. Place the microscope to a position that enables comfortable sitting. 3. Check the completeness of the microscope and switch on the microscope using the main switch located on the base. Note: don’t switch off the microscope in between individual observations, because repeated switching shortens the lifetime of the bulb in the light source. 4. Look at the specimen by the naked eye, before placing it on the stage. Check if it is not upside down and look for the position of the object (in some specimens, the approximate position of the object is marked by a circle). 5. Set the revolving nosepiece to the least magnifying objective (4×). 6. Put the specimen on the stage, fasten it by the specimen holder and place the desired object it into the path of the light beam (above the hole in the middle of the stage). 7. Adjust the distance of eyepieces to fit it for the distance between the centres of your pupils. You should obtain one image (by fusion of images from both eyepieces) that you can observe by both eyes in the same time. Sometimes it needs a bit effort to train you to keep both eyes opened when looking through the oculars. Observation with only one eye leads to eye fatigue, headache or if practiced for a long time, it can cause eye disorders. 8. Adjust the diopter adjustment on the eyepiece, if necessary. 9. To arrange the object into the visual field, change simultaneously the focus (left hand, coarse focus knob) and position of the specimen (right hand, specimen holder movement knobs). 10. Adjust the amount of light by rotating the iris diaphragm and/or by rotating the brightness adjustment knob on the base of the microscope. 11. Look into the eyepieces and find the object first by using the coarse focus knob and then focus it by using the fine focus knob. 12. Observe the object first by using less magnifying objectives and then stepwise switch to more magnifying objectives (for adjusting use only fine adjusting knob). Note: place the object into the centre of the visual field, focus it and adjust the brightness both before and after switching to a more magnifying objective. When you lose the object, you must usually return to a smaller magnification or start again from the beginning. 13. Use a meandering (zigzag) movement and go through the whole space under the cover glass systematically. Start in one corner of cover glass and realize that the image is reversed, so moving the specimen to the left leads actually to moving the image to the right and vice versa. 20 14. Choose the optimal magnification for observation of this particular object, observe it, draw and describe your observation. Note: don’t forget to note the used magnification into your record. 15. After finishing the observation, it is recommended to switch to the smallest magnification, because it facilitates specimen removal 16. If immersion oil was used for the observation (only in case of 100× magnifying objective), clean the immersion objective using a cloth soaked with ethanol or xylene. 17. Switch off the microscope and cover it with dust cover at the end of the lesson (not between the individual observations). Crucial steps for successful observation: 1. placing the object into the centre of the visual field (centring) 2. focusing the object 3. adjusting the brightness (the use of more magnifying objectives requires more light) Note: the amount of light (brightness) can be adjusted in two ways: (1) by rotating the iris diaphragm, (2) by rotating the brightness adjustment knob on the base of the microscope. Troubleshooting: If the observed structure does not change position when you move the slide, then it is an artefact (e.g. dirt on the objective), not really the object on your stage. When it rotates with the rotation of the left or right eyepiece, then it is the dirt in the respective eyepiece. A frequent cause of missing the objects is that you search in a wrong optical plane or that you use excessive brightness. Note: artefacts are undesirable confusing structures not related to the observed object (dirt, drops, bubbles, scrapes). In the table below you can see some of the most common problems, their most probable causes and proposed solving. Problem microscope does not light when switched on coarse focus moves tightly stage moves spontaneously downvards microscope unfocused image, impossible to focus image is cloudy insufficient illumination only a part of the visual field is illuminated the image is not in contrast enough Cause Solving microscope is not plugged in wrong adjustment of the stiffness of the movement wrong adjustment of the stiffness of the movement specimen is upside down the layer of the medium used for sample embedding is too thick plug in the power cord both into the slot of the microscope and into the socket turn the coarse focus knob counterclockwise turn the coarse focus knob clockwise turn the specimen upside down fix the sample objective is dirty (dust, imersed oil) clean the objective (by a piece of cloth or a paper tissue soaked with ethanol) eyepiece is dirty (the dirt moves when rotating the eyepiece) clean the eyepieces move the condenser and adjust the brightness the revolving nosepiece is not in the turn the revolving nosepiece into the proper position proper position (you’ll hear the click) condenser is located too low iris diaphragm is too much opened adjust the brightness (to less light) impossible to find object using small magnification the object (e.g. epithelial cell, objective micrometer or pollen grain) is not contrast enough (it is transparent) adjust the brightness (to less light) and focus on the proper optical plane object is lost when switching to a higher magnification object was not placed in the centre of the visual field return to a lower magnification and place the object into the centre of the visual field 21 TASKS Task 1: Arranging the object in the visual field Permanent preparation (PP) of a letter - “písmeno” Put the permanent preparation with a block letter on the stage of microscope to be able to read the word (not reversed). Draw one asymmetrical letter (a, e, y, k...) using the smaller magnification of the objective (4×). What image can you see in the microscope? TASK 2: Arranging the object using different magnifications (4×, 10×, 40×) PP: insect wing - “křídlo hmyzu” (or letter) Observe the specimen under different magnifications. How does the free working distance, visual field size and resolution change based on magnification used? A B C Fig. 9: Different magnifications of objective (A - 4×, B - 10×, C - 40×) and visual field (insect wing). TASK 3: Centring the object PP: stained wool - “vlna barvená” Place the stained wool (cross of fibers or other characteristic point) in the centre of the visual field under the smallest magnification (4×). Then use the 10× magnifying objective without drifting the object and observe the wool. Can you see some change? (Do the same with 40× magnifying objective). What principle arises from this observation? A B C Fig. 10: Centring the object (stained wool) and different magnification of objectives (A - 4×, B - 10×, C - 40×). TASK 4: The function of iris diaphragm PP: feather - “prachové peří” Observe the feather under different objectives (4×, 10× a 40×) with an opened and closed iris diaphragm. What is the difference and what principle arises from this observation? 22 4.2. Optical planes, measuring the size and thickness of microscopic objects FOR MEASURING THE THICKNESS OF MICROSCOPIC OBJECTS a graded scale located on the fine focus knob is used. The scale is divided into units at 2.5 µm each. Find the microscopic object and focus first on the upper optical plane of the object. Look at the value on the scale. Then focus through the whole depth of the object and stop on the lower optical plane of the object. Read the value on the scale again. The depth (thickness) of the object (in µm) equates the number of units (the difference between the first and second scale reading) multiplied by 2.5 (value of one unit on the scale). 1 unit of the scale = 2.5 m The thickness of the object = 2.5 x number of units MEASURING THE SIZE OF MICROSCOPIC OBJECTS is performed using an eyepiece micrometer. It is a device that can be used instead of one of the eyepieces. It has 1 cm long scale divided into 100 units at 0.1 mm each. The actual size of the units of an eyepiece micrometer at different magnifications has to be first determined by comparison with another precise measuring instrument, an objective micrometer. It is a cover slip (attached to a slide) equipped with 1 mm long scale divided into 100 divisions at 10 µm each. Prior to measuring any object, a micrometric coefficient (i.e. the actual size of one unit of an eyepiece micrometer), has to be determined for each objective. To determine the micrometric coefficient, arrange the objective micrometer into the visual field (the same way as any other specimen). Turn the eyepiece micrometer until it is parallel to the objective micrometer and move the objective micrometer to a position where it will be aligned with the eyepiece micrometer (both the scales start at the same place). Find any two units of the two micrometers that correspond to each other (see Figure 11) and calculate the micrometric coefficient according to the formula below. Start with determining the micrometric coefficient for 4× magnifying objective and subsequently continue with the rest of the objectives including the 100× magnifying one. Note: Do not forget to place the objective micrometer into the centre of the visual field and to focus it before you use a more magnifying objective, and to adjust the brightness. number of units of the objective micrometer x 10 Micrometric coefficient = mircrometrePočet dílků obj. mikrometru × 10 number of units of the eyepiece micrometer 0 10 20 30 40 50 60 70 80 90 100 Eyepiece micrometre Objective micrometer Fig. 11: An example of micrometric coefficient determination (for 4 × magnifying objective). If 100 units of objective micrometer correspond to 40 units of the eyepiece micrometer, the micrometric coefficient is 25 µm (100×10/40). For the respective measuring of microscopic object, arrange the object into the visual field, measure it by the eyepiece micrometer (you do not need the objective micrometer anymore) and multiply the assigned number of units by the micrometric coefficient for the particular objective. As a result, you will get the size of the object in µm. 23 Notes: 1. There is no need to use immersion oil using 100× magnifying objective. 2. Micrometric coefficient has to be determined for each objective of the particular microscope extra and it can’t be used for other objectives. ___________________________________________________________________________ TASKS Task 1: Optical planes and measuring of the thickness of the objects PP: insect wing - “křídlo hmyzu” Rotate the fine focus adjustment knob in one direction and note the first sharp image (a hair), medium (a major branch) and the last (a hair) = upper, middle and lower optical plane. Note which unit of the fine focus adjustment knob corresponds to the first optical plane and which corresponds to the last one (measure it against a fixed point on the coarse knob) and calculate the difference. What is the thickness of the object in μm? A B C Fig. 12: Optical planes (A - upper, B - middle, C - lower). TASK 2: Measuring the length of microscopic objects PP: bird red blood cells - “ptačí krev/krvinky Put the objective micrometer on the stage and observe it using 4×, 10×, 40× and 100× magnifying objectives. The left margins of both scales must be aligned. Read the corresponding numbers of units on both scales where they meet and calculate the micrometric coefficient for each objective. When changing to the next magnification, readjust the centering, brightness, focus and realign the left margins of both scales. Note these coefficients for future measuring of microscopic objects. Measure the length of the red blood cells . Bacteria - coccus Bird erythrocyte Bacteria - bacillus Mammal erythrocyte Pollen grain 10 μm Fig. 13: Comparison of the length of different cells. 24 4.3. Permanent preparations PERMANENT PREPARATIONS are instrumental to multiple observations and can be kept for a long time. The advantage of permanent preparations is the possibility to visualize structures that can’t be observed in native specimens. Preparation of permanent preparation usually comprises fixation (prevents decomposition of biological samples), embedding, sectioning (especially in histological tissue samples that have to be cut into thin sections), staining (by routine or special techniques that give contrast to the sample being examined and help to visualize the differences in cell morphology by selectively staining cells and cellular components) and mounting the object into transparent medium that excludes the air from the sample and thus prevents its oxidation. Some samples (e.g. keratin-based arthropod body parts, hair or feathers) do not require all the preparation steps (fixation, dehydration) and can be simply mounted in between slide and cover slip using gelatine. Preparation of permanent preparation: 1. Obtaining the sample. Material for microscopic specimen preparation can involve tissue samples obtained e.g. by biopsy, or individual cells transferred onto microscopic slide as a smear (blood or bacteriological smears) or as an impression of tissue sample. 2. Fixation is a process by which biological tissues are preserved from decay, either through autolysis or decomposition. Fixation terminates any ongoing biochemical reactions and may also increase the mechanical strength or stability of the treated tissues or cells. Fixation should preserve a sample of biological material as close to its natural state as possible, without altering the sample and introducing artefacts that could interfere with interpretation of cellular ultrastructure. a) Chemical fixation uses various kinds of fixatives for treating different types of cells/tissues to achieve a good preserving effect. Crosslinking fixatives (e.g. formaldehyde, often sold as a 30-40 % saturated aqueous solution under the name formalin) act by creating covalent chemical bonds between proteins in tissue, anchoring soluble proteins to the cytoskeleton and lending additional rigidity to the tissue. Also oxidizing fixatives (such as potassium dichromate or chromic acid) can react with various side chains of proteins and other molecules allowing the formation of crosslink which stabilize tissue structure. Precipitating (or denaturing) fixatives (e.g. ethanol, methanol or acetone) act by reducing the solubility of protein molecules and often by disrupting the hydrophobic interactions which give many proteins their tertiary structure. The precipitation and aggregation of proteins is a very different process from the crosslinking caused by aldehyde fixatives. Acetic acid is a denaturating agent that is sometimes used in combination with the other precipitating fixatives. The alcohols are known to cause shrinkage of tissue during fixation while acetic acid alone is associated with tissue swelling. Combining the two may result in better preservation of tissue morphology. b) Physical fixation can be carried out e.g. by the heat produced by a flame. 3. Embedding and sectioning. The tissue samples have to be cut into thin sections (5 - 20 µm) prior to the staining procedure. Appropriate small pieces of tissue (approx. ½ cm3) are placed in a cassette, dehydrated (using ethanol and xylene), embedded in paraffin and cut on a microtome. Paraffin has to be removed from the samples prior to staining. 4. Staining is used in microscopy to enhance contrast in the microscopic image. Various stains and dyes can be used to highlight different structures in tissues or cells for viewing. Stains may be used to define and examine tissues (e.g. muscle fibers or connective tissue), cell populations (e. g. different blood cells) or organelles within individual cells. The staining techniques described below are used in light microscopy. Different staining techniques are used for sample preparation in electron microscopy 25 (see Chapter 3.3.). Fluorescent dyes (e.g. a fluorescent nuclear stain DAPI, or ethidium bromide) can be used for special purposes, e.g. apoptosis detection. According to vital state of stained cells, we distinguish: a) Vital staining (in vivo staining) is the process of dyeing living cells or tissues. If certain cells or their structures take on contrasting color(s), their morphology or position within a cell or tissue can be seen and studied. The usual purpose is to observe cytological details that might otherwise not be apparent (e.g. vacuole staining by neutral red or mitochondria staining by Janus green). Vital staining can reveal where certain chemicals or specific chemical reactions take place within cells or tissues. b) Postvital staining (in vitro staining) involves coloring cells or cell structures that are no longer living. Several dyes are often combined to reveal more details and features than a single stain alone. Combined with specific protocols for fixation and sample preparation, scientists and physicians can use these standard techniques as consistent, repeatable diagnostic tools (e.g. Gram staining in bacteriology or May-Grünwald and Giemsa-Romanowski staining in haematology). Different dyes can be used to stain different structures of a cell or tissue. In general, acid dyes are used to stain cytoplasm and basic dyes stain nuclei. Some of the most common biological stains are listed below. Crystal violet stains cell walls purple. It is an important component in Gram staining. Eosin is most often used as a counterstain to haematoxylin, giving a pink or red color to cytoplasmic material, cell membranes, and some extracellular structures. It also gives a strong red color to red blood cells. Carbolfuchsin may be used to stain collagen, smooth muscle, or mitochondria. Haematoxylin stains nuclei blue-violet or brown. It is most often used with eosin in haematoxylin and eosin staining, one of the most common procedures in histology. Iodine (component of Lugol solution) is used as an indicator for starch. When starch is mixed with iodine in solution, an intensely dark blue color develops, representing a starch/iodine complex. Iodine is also one component of the Gram staining. Methylene blue is used to stain cell nuclei. Neutral red (or toluylene red) stains nuclei red. It is usually used as a counterstain in combination with other dyes. Different staining techniques were developed, that combine more different dyes to reach the desired effect. Some of the most common staining procedures are listed below: Gram staining is used to determine gram status to classify bacteria. It is based on the differences in composition of bacterial cell walls. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a carbolfuchsin or safranin counterstain to mark all bacteria. Gram-positive (G+) bacteria stain dark blue or violet, because their cell wall is typically rich in peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative (G-) bacteria that appear red or pink after Gram staining. Haematoxylin and eosin staining protocol is used frequently in histology to examine thin sections of tissue. Haematoxylin stains cell nuclei blue, while eosin stains cytoplasm, connective tissue and other extracellular substances pink or red. Eosin is strongly absorbed by red blood cells, coloring them bright red. Romanowski staining is based on a combination of eosinate (chemically reduced eosin) and methylene blue (sometimes with its oxidation products azure A and azure B). Common variants include Wright’s stain, Jenner’s stain, Leishman stain and Giemsa stain. They are used to examine blood or bone marrow samples. They are often used for inspection of blood cells because different types of leukocytes can be 26 readily distinguished, and are also suited to examine blood to detect blood-borne parasites. Sudan staining uses Sudan dyes (Sudan III, Sudan IV, Oil Red O or Sudan Black B) to stain sudanophilic substances such as lipids (orange color). 5. Dehydratation. As most of the mounting media do not mix with water, the water in the specimen has to be replaced by alcohol or other solvent prior to mounting procedure. 6. Mounting. For mounting microscopic object in between slide and cover glass, various media are used, providing optimal optical environment for sample observation and enclosing the microscopic object, thus protecting it and enabling long time storage. Liquid media (e.g. glycerol) are rarely used. Solidifying media are liquid in the time of specimen preparation and solidify later on. Canada balsam is one of the best known mounting media. It is a turpentine made from the resin of the balsam fir tree (Abies balsamea), it has high optical quality and refractive index (n=1.55) very close to that of glass. Glycerol gelatine, gum Arabic (taken from two species of acacia trees) or synthetic resins are also frequently used. 7. Description and storage. Complete permanent preaprations should be inscribed with the object type (cell, tissue, species) and by date and method of preparation. They should be stored in the dark and protected from dust and mechanical damage. ___________________________________________________________________________ TASKS TASK 1: Blood smear Drop the anticoagulant-treated blood onto the slide glass. Use a skewed glass at an angle of 45° to smear the blood drop on the slide glass by pulling the drop behind the skewed glass. Otherwise you will damage and change the shape of the erythrocytes (they are extremely fragile). Please avoid these mistakes: small or too large drop of blood, oily slide glass, unequal smear, accumulation of blood at the end of smear. TASK 2: Blood smear and „Panoptic” staining after Pappenheim (MayGrünwald, Giemsa-Romanowski) Prepare the blood smear. Apply the May-Grünwald (5 min.); add distilled water (5 min.) and pour off dye. Apply the Giemsa-Romanowski (10 min.), pour off dye and rinse it with distilled water. Dry it by the careful by using a filtration paper. Observe the red blood cells in the thin layer, measure the diameter of a mammal erythrocyte. Compare the mammal blood smear with a permanent preparation of a bird blood smear. Measure the size of bird erythrocyte. A D B E C Fig. 14: Procedure: A – put drop of blood on the slide, B – attach a glass to the drop at an angle of 45o, C – make blood smear. Comparison of erythrocytes: D – bird (oval, with nucleus), E – mammal (round, without nucleus). 27 TASK 3: A preparation of a permanent preparation (glycerol-gelatine). Drop heated glycerol-gelatine, melted in a water bath, on the slide. Place a rabbit hair into the gelatine and cover it by a cover glass. Cuticle Corneous cells filled with air Fig. 15: Rabbit hair. 4.4. Native preparation, phase contrast NATIVE PREPARATIONS are samples obtained from living objects without fixation. They can’t be stored for a long time. Such samples are usually observed in a liquid medium in which they occur in natural conditions (water, blood serum, plasma, or culture medium), or in isotonic solutions such as physiological solution or PBS (phosphate buffered saline). Some objects (e.g. cutaneous structures arising from the epidermis, such as hair or feathers, or chitinbased arthropod body parts, such as insect wings) can be observed by placing them between the slide and cover slip, without a liquid medium. Observation of fast-moving organisms can be facilitated by the addition of some viscous substance (e.g. glycerol, gelatine, or gum Arabic) or small amounts of narcotics (ether, or chlorophorm) to the sample and by removing excessive liquid or just by increasing the pressure on the cover slide by a finger. The microscopic sample should be thin and transparent, therefore it is recommended to appropriately dilute liquid samples and to compress or tease tissue samples. Vital (or in vivo) staining techniques are used to stain native preparations. PHASE CONTRAST MICROSOPY is suitable for observation of native, non-stained specimens (preferably living objects) that are much more obvious in the phase contrast. Amoebae, for example, look like vague outlines in a bright field, but show a great deal of detail in phase. The phase contrast technique gives contrast to some cell structures (e.g. cilia and flagella) that are otherwise hardly visible. For phase contrast microscopy a normal light microscope is used, but specially prepared for this purpose. You need the phase contrast objectives (10× and/or 40× magnifying, marked with PL and black rubber ring) and corresponding phase contrast condensers. Prior to observation using phase contrast, the light path must be aligned. The condenser ring diaphragm image has to be aligned (centred) with an objective ring diaphragm. These rings are visualized using the phase microscope, a device that is inserted into the microscope tube in place of the eyepiece and when the centring has been done, it is replaced by the eyepiece again. Centring of the diaphragms involves sliding the phase condenser into the light path by rotating the adjustment screws on the phase condenser. A yellow-green filter, that transmits light with a wavelength of approximately 540 nm, can be used to increase the contrast of the image. Setting up a microscope for phase contrast microscopy: select the phase objective (10× or 40× magnifying, marked with black rubber ring) and the corresponding phase condenser remove the blue filter from the lower part of the condenser and replace it with the phase condenser 28 open the condenser diaphragm and move the stage into its highest position replace one eyepiece with an phase microscope focus the phase microscope by lifting its inner part and fix it by a screw in the focused position centre (align) the ring-shaped phase condenser diaphragm to that of the phase objective by rotating the two silver adjustment screws on the phase condenser replace the phase microscope by an eyepiece again you can place a yellow-green filter on the window lens Phase objective diaphragm Phase condenser diaphragm Phase condenser A B Fig. 16: Phase condenser and phase objective diaphragms: A – non-centred, B – centred. __________________________________________________________________________________________ TASKS TASK 1: Ciliates (Infusoria) Native preparation (NP): hay infusion (or ciliates from soil) Take a water drop from the hay infusion surface, place it on a slide glass and cover with a cover glass so as not to form air bubbles. Observe the fast moving protozoans, especially ciliates, flagellates and also bacteria, vinegar worms or flatworms. Mistakes that can be made: too much water results in vibrations or drifting of the objects away from visual field, a wet lower side of the slide (and thus wet stage) leads to the impaired control of the specimen movement; lack of water results in air bubbles forming and drying of the sample. Infusoria are named after hay infusion (water, hay, decomposing plants and leaves, soil) in which they can be found. In hay there are cysts of infusorians that give rise to active ciliates. Spheric, spiral and baculiform bacteria Colpidium colpoda (ciliate) Flagellate Vinegar worm (nematode) Philodina - Rotifer Stylonychia mytilus (ciliate) Flatworm Fig. 17: Soil organisms. 29 TASK 2: Mould – kingdom Fungi NP or PP: mould (Rhizopus sp., Aspergillus sp. - kingdom Fungi) – “spory plísní” Prepare a native preparation from bread covered with mold and observe hyphae (with cell walls and nuclei) and spores of mould. Spores Hyphae Nucleus Cell wall Fig. 18: Hyphae and spores of mold. TASK 4: Digestion and contractile vacuoles of protozoans NP: hay infusion - water surface with ciliates (infusorians) and numerous bacteria Take a water drop from the hay infusion surface; put it on a slide glass, stain with 0.1 % neutral red (possibly causing death of protozoans after some time) and cover with a cover glass. Observe the contractile vacuole (for osmoregulation), colorless round structures near a pole of a ciliate alternately increasing size and disappearing after emptying to the outer environment. Fast motion of ciliates can be slowed down by soaking off the excessive water using a filter paper. TASK 5: Digestive vacuoles PP: digestive vacuoles containing charcoal – “potravní vakuoly” The ciliate Paramecium (Paramecium sp.) was cultured in a medium containing charcoal. The digestive vacuoles are stained black due to charcoal contained in them. Digestive vacuoles Contractile vacuoles A B Fig. 19: Ciliate with digestive and contractile vacuoles. A – native staining, B – stained with charcoal. TASK 6: Phase contrast microscopy NP: ciliates or pollen grains Prepare microscope for observation in the phase contrast and then observe ciliates (or pollen grains). Observation in phase contrast increases contrast of colourless structures. 30 5. Prokaryotes and immersion microscopy 5.1. Prokaryotes Prokaryotes evolved earlier then eukaryotes, thus the composition of prokaryotic cell is much simpler. Prokaryotes lack cell nucleus (karyon) and any other membrane-bound organelles (mitochondria, plastids, Golgi apparatus, endoplasmic reticulum, etc.). Most prokaryotes are unicellular. The average size of prokaryotic cell is from 1 to 10 μm. Nucleoid (prokaryotic equivalent of nucleus) consists of one circular DNA molecule (in complex with proteins) and lacks a nuclear envelope. Prokaryotes have 70S ribosomes each consisting of a small (30S) and a large (50S) subunit. Prokaryotes reproduce through asexual reproduction, usually by binary fission (which differs markedly from mitosis in eukaryotes). The prokaryotes are divided into two domains: the bacteria and the archaea. 5.1.1. Domain Bacteria Bacteria are unicellular organisms that are characterized by a prokaryotic cell type. Most of them have cell walls (except in the members of genus Mycoplasma). Bacterial cell walls differ from those of plants and fungi, which are made of cellulose and chitin, respectively. They are made of peptidoglycan murein. In the bacterial cell membrane, the fatty acids are linked to glycerol by ester bonds (like in eukaryotes). Bacterial genes do not contain introns (non-coding DNA regions in a gene that are not translated into proteins), and they are often organised into operons (transcriptional units that contain more structural genes under the control of a single promoter and are transcribed into one polycistronic mRNA, a single mRNA molecule that codes for more than one protein). Besides the nucleoid, bacteria may also contain plasmids, small extrachromosomal DNA molecules that may contain e.g. genes for antibiotic resistance. In prokaryotes, N-formylmethionine is a starting amino-acid in the proteosynthesis (unlike in eukaryotes, where protein synthesis starts with methionine). Bacteria reproduce asexually (by binary fission or by budding). Being asexual organisms, bacteria inherit identical copies of their parent's genes and so the evolution in bacteria is limited to the selection based on recombination or mutations of bacterial DNA. Some bacteria are able to transfer genetic material between cells. This can occur in three main ways: 1) Transformation is uptake of exogenous DNA from the environment. 2) Transduction is gene transfer and integration into the genome by a bacteriophage. 3) Conjugation is process in which DNA is transferred through direct contact between cells occurring in natural conditions and enabling e.g. transfer of genes responsible for antibiotic resistance between different pathogens. Bacteria exhibit an extremely wide variety of metabolic types. They can be both autotrophic (photoautotrophic or chemoautotrophic) and heterotrophic (photoheterotrophic or chemoheterotrophic). Photosynthetic bacteria do not have chloroplasts, thus photosynthesis usually takes place in thylakoid structures on the cell membrane. They use different types of bacteriochlorophylls (c, d, or e) in addition to chlorophyll a. Except of the call wall, many bacteria possess extracellular structures, such as flagella, fimbriae or pili. Flagella are rigid protein structures (about 20 nm in diameter and up to 20 µm in length), that are used for locomotion. They are made up of the protein called flagellin and the energy for their rotating movement is released by transfer of ions across the cell membrane. Fimbriae are fine protein filaments (210 nm in diameter and several µm long) that are distributed over the surface of the cell. They are believed to be involved in attachment to solid surfaces or to other cells and are essential for the virulence of some bacterial pathogens. Pili are cellular appendages that are used to 31 transfer genetic material between bacterial cells during conjugation. Many bacteria also produce capsules or slime layers to surround and protect their cells. Types of bacteria: 1. According to the shape: a) Round (cocci): in pairs of two joined cells (diplococci), in chains (streptococci), tetrads (tetracocci), or group together in clusters (staphylococci). b) Rod-shaped (“rods”, or bacilli): in pairs (diplobacilli), chains (streptobacilli), or in palisades. Members of the genus Bacillus produce oval endospores under stressful environmental conditions. These endospores can be placed at one end or in the middle of the cell and they can make the cell bulge. c) Slightly curved or comma-shaped (vibrio). d) Spiral-shaped (spirilla). e) Tightly coiled (spirochaetes). f) Filamentous - e.g. Actinobacteria. 2. According to degree of flagellation: a) Atrichous - lack flagella. b) Monotrichous - have a single flagellum. c) Lophotrichous - have two or more flagella at one polar attachment point. d) Amphitrichous - have two flagella, one at either end of a cell. e) Peritrichous - have a large number of flagella in many places of the cell surface. 3. According to cell wall composition: a) Gram-negative (G-) bacteria have a relatively thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. G - bacteria do not retain crystal violet dye when washed in a decolorizing solution (ethanol) during Gram staining procedure. A counterstain (commonly carbolfuchsin or safranin) used after the crystal violet stains G- bacteria red or pink. Medically relevant G- bacteria include e.g. the coccus Neisseria gonorrhoeae, which cause a sexually transmitted disease gonorrhoea, or bacilli that cause respiratory (Haemophilus influenzae, Klebsiella pneumoniae), urinary (Escherichia coli, Proteus mirabilis), or gastrointestinal problems (Helicobacter pylori, Salmonella typhi). b) Gram-positive (G+) bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids. These bacteria are stained dark blue or violet by crystal violet dye during Gram staining. This group includes well-known genera, such as Staphylococcus, Streptococcus, Enterococcus, Bacillus, Listeria, or Clostridium. c) Gram-variable bacteria stain irregularly or inconsistently by Gram staining. For example Mycobacterium tuberculosis, or Coxiella burnetii are sometimes referred to react as Gram-variable. d) Gram neutral bacteria stain very poorly or not at all using Gram staining (e.g. members of the genus Mycoplasma, that lack cell wall). Some important groups of bacteria: 1. Nitrifying bacteria oxidize ammonia into nitrites that are subsequently oxidized into nitrates. Ammonia oxidizing bacteria (e.g. Nitrosomonas or Nitrococcus) play an important role in the nitrogen cycle in soil. Rhizobia form another group of soil bacteria that fix nitrogen. They live in symbiosis with legumes (plants belonging to the family Fabaceae, with the well-known members, such as clover, beans, lentils, alfalfa, lupines or peanuts). The rhizobia are not able to fix nitrogen independently and require to be established inside root nodules of legume plants. 32 2. Cyanobacteria (also known as „blue-green algae“, or Cyanophyta) is a phylum of bacteria that obtain energy through photosynthesis, that takes place on thylakoid membranes very similar to those of chloroplasts. In cyanobacteria, phycobiliproteins capture light energy which is then passed on to bacteriochlorophylls during photosynthesis. Phycobiliproteins are responsible for the blue-green pigmentation of most cyanobacteria. Variations to cyanobacteria coloration are mainly due to carotenoids and phycoerythrins which give the cells the red-brownish color. Cyanobacteria inhabit almost every conceivable environment, from bare rock and soil to both salt and fresh waters, thus being important components of plankton and benthos. The name of the Red Sea is derived from the color changes observed in its waters that are occasionally populated by Trichodesmium erythraeum, which, upon dying off, gives the sea a reddish color. 3. Symbiotic bacteria live in symbiosis with other organisms. In digestive systems of many organisms, symbiotic bacteria help break down food or produce vitamins. Mammals do not have the ability to break down cellulose directly, thus proventriculus flora of some ruminants (cattle and sheep) contains symbiotic anaerobic bacteria (e.g. Cellulomonas) that produce enzymes to break down cellulose. Many herbivores (like rabbits and horses) have large ceca of the large intestine that host symbiotic bacteria. Many bacterial genera are also able to produce vitamins in the gut (e.g. vitamin B12 that cannot be synthesized by animals). Symbiotic bacteria also play an important role in the lives of many insects. Many termite species have symbiotic bacteria (and also protozoans) in the gut, enabling them to digest cellulose. 4. Pathogenic bacteria cause diseases of both humans and animals (and also plants), for example: tetanus (Clostridium tetani), cholera (Vibrio cholerae), typhoid fever (Salmonella enterica serovar Typhi), diphtheria (Corynebacterium diphtheriae), tuberculosis (Mycobacterium tuberculosis), and peptic ulcer disease (Helicobacter pylori). Some bacteria can infect both humans and animals (e.g. Bacillus anthracis, the causative agent of anthrax, or Yersinia pestis, carried by rodents and causing plague). Animal diseases that can be transmitted to humans are known as zoonoses. Some of the bacteria can be transmitted by arthropod vectors, e.g. Borrelia burgdorferi, the causing agent of Lyme disease, is transmitted by Ixodes ticks. Some bacterial species (e.g. Mycobacterium avium or Pseudomonas aeruginosa) cause disease mainly in individuals suffering from immunosuppression (opportunistic pathogens). Bacterial infections may be treated with different antibiotics (bacteriocidal or bacteriostatic drugs) that differ in their mode of action and in spectrum of bacteria. Antibiotics always inhibit a process that is different in the pathogen from that found in the host (selective toxicity). Some of the bacterial infections can be prevented by vaccination. Bacterial infections can be prevented by antiseptic measures such as sterilization or disinfection. 5.1.2. Domain Archaea Archaea are unicellular organisms that are characterized by prokaryotic cell type and reproduce asexually (by binary fission or by budding). Like bacteria, archaea have no internal membranes and their genome is formed by single DNA molecule (nucleoid). Archaea are quite similar to bacteria in size and shape, although a few archaea have very unusual (flat or square) shapes. Despite this visual similarity to bacteria, archaea possess several features (including archaean RNA polymerase or ribosomes) that are more closely related to their equivalents in eukaryotes. Like in eukaryotes, initiator tRNA in archaeal translation carries methionine; most of archaeal genes encoding tRNA and rRNA and few of their structural 33 genes contain introns. Archaeal genes are often organised into operons like bacterial genes. Other aspects of archaean biochemistry are unique, such as the lipids in cell membranes, that contain ether linkages between the glycerol backbone and the fatty acids (unlike bacteria and eukaryotes, where fatty acids are linked to glycerol by ester bonds), or the composition of their cell wall, that, in contrast to bacteria, usually lacks peptidoglycan but can contain pseudopeptidoglycan (pseudomurein). Members of genera Thermoplasma and Ferroplasma lack cell walls. Spores are formed by both bacteria and eukaryotes, but are not formed by any of the known archaea. Archaea are phototrophs, lithotrophs or organotrophs (obligatory or facultative). Archaea exist in a broad range of habitats, and are a major part of global ecosystems. They may contribute up to 20 % of the total biomass on Earth and form a major part of oceanic life. Multiple archaeans are extremophiles (halophiles, thermophiles, alkaliphiles or acidophiles): some archaea survive high temperatures, often above 100°C, others are found in highly saline (up to 23 % NaCl), acidic, or alkaline water. However, other archaea are mesophiles that grow in much milder conditions, in marshland, sewage, the oceans or in soil. 5.2. Observation using immersion objective Immersion objectives enable bigger magnification (and thus higher resolution) compared to dry objectives. The object has to be found and focused using less magnifying objectives (subsequently from 4× to 40× magnifying objective) prior to observation using an immersion objective. After focusing and centring the appropriate area of the sample by 40× magnifying objective, turn the revolving nosepiece into an intermediate position between 40× and 100× magnifying objectives (if the revolving nosepiece does not remain in the intermediate position, it is necessary to hold it with your hand). Put a drop of immersion oil on the slide (into the path of the passing light), turn the revolving nosepiece, so that 100× magnifying objective immerses into the drop of immersion oil. Move the stage with fine adjusting knob to the higher position close to 100× 100× the objective. Then move the stage down by rotating with fine focus adjustment knob to focus object (see Figure). After finishing your observation, clean the lens of Immersion immersion objective (and permanent oil preparation) using a cloth soaked with ethanol or xylen. ___________________________________________________________________________ TASKS TASK 1: Bacteriological smear NP: bacteria (G+ and G- bacteria), PP: stained bacteria Sterilize the bacteriological loop using the gas burner flame and let the loop cool off (not to kill the bacteria). Take a colony of bacteria from the surface of the agar medium in a Petri dish with the bacteriological loop and make a thin smear in a droplet of water on a slide. Let it dry and fix it over the flame (three times). Gram-staining: Apply the crystal violet (the primary stain) on the slide for 3 min. Rinse it with distilled water and apply the Lugol solution (iodine solution) for 2 min. Rinse it and add the ethanol (decolorizer) until the stain stops being washed out. Apply the carbolfuchsin (counterstain) for 1.5 min. 34 Rinse it with distilled water and dry with filter paper. Observe and draw G+ (purplish-blue) and G- (red) bacteria under immersion objective. A B C A Fig. 20: Bacteriological smear: A – transfer of bacteria by bacteriological loop, B – homogenisation of bacteria with water, C – bacteriological smear. TASK 2: Smear impression of human tongue mucosa Take a clean slide (from a box labelled “otisk jazyka”) and anneal it in a gas burner flame. Let it cool off and then impress the surface of your tongue (after roughing it with your teeth) on the slide. Let the slide dry and then fix it above the flame. Perform Giemsa-Romanowski staining for 10 min. Rinse the dye with distilled water and dry the slide gently using a filtration paper. Observe the stained epithelium cells (polygonal shape, nucleus) and bacteria under immersion objective with immersion oil and measure their size. Epithelium cell Bacteria Fig. 21: Epithelium cells of tongue and bacteria. TASK 3: Cyanobacteria “Blue-green algae” (Cyanobacteria; genus Oscillatoria) Observe the structure of cyanobacteria and compare it with cells of green algae or diatoms (frustules), which can be also present in the sample. Just by the preparing of the sample, you can observe oscillatory motion of cyanobacteria. 35 Cyanobacteria Green algae Diatom Fig. 22: Comparation of cyanobacteria with green algae and diatom. 36 6. Chemical composition of bioplasm 6.1. Elements All living organisms are composed of chemical substances (both the inorganic and organic). These molecules consist of elements that appear in roughly the same proportion as in all organisms (differing significantly from their proportion of appearance in nonliving (i.e. abiotic) nature. Hydrogen, oxygen, nitrogen, carbon, phosphorus, and sulfur make up more than 99 % of the mass of all living cells. Elements that occur in living systems are called biogenic elements. MAJOR BIOGENIC ELEMENTS include C, H, O, N, S, P, K, Na, Cl, Ca, Mg and Fe. The most important are C, H, O, N, S and P because they are the basic components of biopolymers. In nature, the main source of carbon (C) is CO2. Hydrogen (H) and oxygen (O) are bound together to form H2O, the basic substance in the cells. Nitrogen N) is the basic component of nucleic acids and proteins. Sulfur (S) is a component of some amino acids. Phosphorus (P) is a part of nucleic acids and ATP (adenosintriphosphate) whose macroergic bonds are used for energy storage. Calcium (Ca) serves as the second messenger in cell signalling and enables muscle contraction. MINOR BIOGENIC ELEMENTS (TRACE ELEMENTS OR MICRONUTRIENTS) are chemical elements that are needed in minute quantities for the proper growth, development, and physiology of an organism. They have well-established functions (e.g. as cofactors of enzymes). Trace elements include heavy metals (cobalt, copper, manganese, and molybdenum) and also other elements, such as chromium, iodine, selenium, zinc, silicium, boron and other. Higher amount of heavy metals can accumulate in body and lead to a toxic effect. 6.2. Chemical compounds Among the most important molecules, living cells contain water (70 %), proteins (15 %), nucleic acids (7 %), polysaccharides (2 %) and lipids (2 %). WATER normally accounts for approximately 70 % of the total wet-weight of the cells with the exception of some spores and seeds that contain little water due to adaptation for survival in unfavourable conditions for extended periods of time. Most of the essential biochemical reactions take place in aqueous environment. Water also helps to keep the homeostasis of an organism. Due to its heat-carrying capacity it helps to maintain a constant temperature and it is important for the acid - base equilibrium and for keeping osmotic pressure in the cells. PROTEINS are made of monomer amino acids (AA) that are arranged in a linear chain and joined together by covalent peptide bonds between the carboxyl and amino groups of adjacent amino acid residues, forming a polypeptide chain with aminoacyl (NH2 group) and carboxyl ending (COOH group). H NH2 C COOH R Fig. 23: General chemical formula of an amino acid (NH2 – amino group, COOH – carboxyl group, R – side chain). 37 Amino acids are either used to synthesize proteins and other biomolecules or oxidized to urea and carbon dioxide as a source of energy. Proteins in living cells are made of 20 different standard amino acids (plus selenocysteine that is not coded for directly in the genetic code, but is encoded in a special way by a UGA codon which is normally a stop codon). Aminoacids are designated by three or one letter codes e.g. alanine (Ala, A), asparagine (Asn, N), glycine (Gly, G), valine (Val, V). The number of amino acids in the chain of common proteins is about 300 amino acids. Main protein functions: Structural: proteins are part of cell structures such as cytoskeleton, chromosomes, ribosomes, biomembranes or extracellular matrix. Catalytic: as enzymes, they catalyze most of the biochemical reactions. They increase the rates of the reactions by lowering their activation energy. Informative: they take part in cell signalling and signal transduction. Some proteins, such as insulin, are extracellular signal transmitters. Other proteins are membrane receptors whose main function is to bind a signalling molecule and to induce a biochemical response in the cell. Transmembrane proteins can also serve as ligand transport proteins that alter the permeability of the cell membrane to small molecules and ions (e.g. potassium or sodium channels). NUCLEIC ACIDS (NA) are made of monomer nucleotides, the sequence of which determines the primary NA structure. Nucleotides are structural units of NAs and consist of three joined structures: a nitrogenous base, a sugar (pentose) and a phosphate group. Nucleotides are usually divided into two groups (purines and pyrimidines) based on the structure of the nitrogenous base. Purine bases are adenine (A) and guanine (G), pyrimidine bases are cytosine (C), thymine (T) and uracil (U). Base pairing follows the rule of complementarity: purine base pairs with pyrimidine base by two or three hydrogen bonds (A=T, A=U and C≡G). Deoxyribonucleic acid (DNA) consists of backbone made of sugar molecules (deoxyribose) and phosphate groups joined by ester bonds (5´ending with phosphate and 3´ending with sugar). One of four bases (adenine, guanine, cytosine and thymine) is attached to each sugar by glycosidic bond. The secondary DNA structure is usually right handed double helix with the two strands running in opposite (anti-parallel) directions to each other. DNA secondary structure was described by J. Watson and F. Crick in 1953. Within cells, DNA is organised into structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication (S phase of cell cycle). Eukaryotic organisms (animals, plants, fungi, and protists) store their DNA inside the cell nucleus, while in prokaryotes (bacteria and archaea) it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organise DNA. These compact structures guide the interactions between DNA and other proteins, helping control the DNA transcription. Ribonucleic acid (RNA) consists of a phosphate, a ribose sugar and nitrogenous bases adenine, guanine, cytosine and uracil. RNA is usually single-stranded (some viruses possess double stranded RNA in their genomes). Types of RNA: 1. Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes. It is coded so that every three nucleotides (a codon) correspond to one amino acid. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to a mature mRNA by removing introns (non-coding sequences of gene). The mRNA is then exported from the nucleus to 38 the cytoplasm, where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA. 2. Transfer RNA (tRNA) is a small RNA chain that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment and an anticodon region that binds to a specific sequence on the mRNA chain through hydrogen bonding. 3. Ribosomal RNA (rRNA) is the catalytic Binding site for component of the ribosomes. Eukaryotic amino acid ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. Three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized elsewhere. In the cytoplasm, ribosomal RNA and proteins combine to form a nucleoprotein particle called ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes are usually attached Anticodon to a single mRNA, forming the so-called polysome. Fig. 24: tRNA structure – “clover leaf”. Main nucleic acid functions: Deoxyribonucleic acid is often compared to a set of blueprints, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The main role of DNA molecules is the long-term storage of informations. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. In some viruses, RNA exerts these functions instead. Ribonucleic acids play several important roles in the processes of transcribing genetic information from DNA into proteins. RNA acts as a messenger between DNA and the protein synthesis complexes known as ribosomes, forms vital portions of ribosomes, and serves as an essential carrier molecule for AA to be used in protein synthesis. POLYSACCHARIDES are composed of monosaccharides with general formula (CH2O)n. Monosaccharides are linked together by covalent glycosidic bond to form disaccharides, oligosaccharides and polysaccharides. Main saccharide functions: Energy storage and metabolism: glucose and glycogen (in animals) and starch (in plants). Structural: cellulose and chitin are the main component of cell wall in plants and in fungi, respectively. In arthropods, chitin forms much of the exoskeleton. LIPIDS are made of fatty acids and glycerol. Main lipid functions: Structural: phospholipids are the main component of cell membranes Energy storage and metabolism: triacylglycerols stored in adipose tissue are a major form of energy storage in animals Signalling: steroid hormones, such as estrogene, testosterone or cortisol, which are essential for reproduction, metabolism or blood pressure control, are lipids. Recently, several different lipid categories have been identified as signalling molecules and cellular messengers that are important parts of the cell signalling. 39 Vitamin metabolism: the "fat-soluble" vitamins (A, D, E and K) are essential nutrients that are stored in the liver and fatty tissues. ___________________________________________________________________________ TASKS TASK 1: Proof of starch Press a piece of potato against a slide (or scrape off some juice from a cut half of potato), add a drop of water and cover with the cover glass. Observe starch grains consisting of concentric layers. Add Lugol’s solution (KI + I2 + H2O) and observe the blue color of the stained starch. Fig. 25: Starch grains. TASK 2: Proof of enzyme – amylase Flush your mouth with water (saliva contains enzyme amylase, which splits starch into simple sugars), filtrate it using a funnel, wet filtration paper and beaker. Prepare six tubes. The first one will contain 1 ml of saliva (concentrated amylase), second to fifth tubes will contain 1 ml of decreasing concentrations (1/2, 1/4, 1/8, 1/16) of amylase prepared by diluting saliva with physiological solution (0.9 % NaCl). The sixth tube will be a control containing 1 ml of physiological solution. Add 2 ml of starch to each tube including the control tube. Incubate the tubes at 40°C in a water bath for 5 min. Add Lugol’s solution to the control tube to permanent blue color and add the same amount to all other tubes. Evaluate the color of all tubes and explain why the color changed. Dilution of amylase Concentrated amylase 1 ml amylase 1 ml amylase 1 1/2 Control transfer 1 ml 1/4 1/8 1 ml NaCl 40 1/16 TASK 3: Proof of DNA NP: internal epidermis of onion Put an internal epidermis of onion onto the slide, fix it with 1 % acetic acid for few minutes, then stain it with 1 % methyl-green dye for 5 minutes and cover it with the cover glass. TASK 4: Proof of fat PP: histological section of fatty liver tissue stained by Sudan III red dye “lipidy” Observe the orange-red lipid vacuoles inside the liver cells. Lipid vacuoles Cell nuclei containing DNA Nucleus Fig. 26: Cell nuclei containing DNA stained blue-green. Fig. 27: Lipid vacuoles in liver cells. TASK 5: Proof of protein (after Heller) Pour 2 ml of nitric acid into the tube and slowly add egg white (the two liquids must not mix). You can observe a white ring of denatured protein between both layers. TASK 6: Proof of water Anchor a potato cube (about 1 cm3) at the bottom of a tube with a skewer and pour 2 ml of glycerol into a tube. Observe the water escaping from the potato into glycerol (hypertonic surrounding) and rising through it. Skewer Egg white Glycerol White ring of denatured protein Water HNO3 Potato Fig. 28: Proof of protein (ater Heller). Fig. 29: Proof of water. 41 7. Non-cellular life Non-cellular life is life that exists without cells. This term presumes the phylogenetic classification of viruses as life forms. Besides viruses, other forms might also be included, like viroids, satellites (virusoides) or prions. Most of them are sub-microscopic infectious agents (usually with much simpler organisation than a cell) which are not able to grow or reproduce outside a host cell. VIRUSES are sub-microscopic infectious agents that are unable to grow and reproduce outside a host cell. Viruses infect all cellular life, though not all viruses cause diseases. Many viruses reproduce without causing any obvious harm to the infected organism. Some viruses (such as HIV) can cause life-long or chronic infections, and the viruses continue to replicate in the body despite the host’s defence mechanisms. Most of the viruses are about 100 times smaller than bacteria with diameter between 10 and 300 nm (Mimivirus found inside the amoeba measures 400 nm). Viruses spread in many different ways: plant viruses are often transmitted from plant to plant by insects, while animal viruses can be carried by bloodsucking insects. These disease-bearing organisms are known as vectors. Influenza viruses are spread by coughing and sneezing, and others such as norovirus, are transmitted by the faecal-oral route, when they contaminate hands, food or water. HIV is one of several sexually transmitted viruses. Virion is a term used to refer to a single complete infective viral particle. Virions consist of two or three parts: all viruses consist of nucleic acid (DNA or RNA), that is surrounded by a protective coat of protein called a capsid. Capsid is formed from identical protein subunits called capsomers. Some viruses have a lipid envelope derived from the host cell membrane. Enveloped viruses (e.g. influenza virus or HIV) envelope themselves in a modified form of one of the cell membranes, either the outer membrane surrounding an infected host cell, or internal membranes such as nuclear membrane or endoplasmic reticulum, thus gaining an outer lipid bilayer known as a viral envelope. This membrane is covered with proteins coded for by both the viral and host genomes. Bacteriophages (or simply phages) are viruses that infect bacteria. Capsids of most of the phages have complex structure, consisting of an icosahedral head (containing nucleic acid – DNA or RNA) bound to a helical tail which may have a hexagonal base plate with protruding protein tail fibers (for attachment to a host cell). Bacteriophages can be used in phage therapy directed against specific bacteria. Virus classification is based mainly on phenotypic characteristics (capsid morphology, nucleic acid type, envelope, etc.), on the mode of replication, host organisms, and on the type of disease they cause. Virus taxonomy involves various classification systems (among the most frequently used are Baltimore classification and ICTV classification). Virus classification and taxonomy is based on: 1) Morphology and size of the virion: helical, icosahedral, complex viruses 2) Presence of the envelope: enveloped and naked viruses 3) Nucleic acid type and structure and mechanism of mRNA production: a) NA type: RNA viruses, DNA viruses b) Number and shape of NA molecules: ssRNA (single stranded RNA), dsRNA (double stranded RNA), ssDNA, dsDNA, linear or circular NA c) NA strand polarity: (+)RNA (plus RNA with the same polarity as mRNA), (-)RNA (minus RNA), (+)DNA, (-)DNA 42 4) Host specificity: e.g. animal, vertebrate, invertebrate and plant viruses, bacteriophages and cyanophages 5) Affinity to tissues: e. g. respiratory, enteric, hepatic and sexually transmitted viruses Viral reproduction in host cells: LYTIC CYCLE is way of viral reproduction that results in the destruction of the host cell: 1. Attachment: virus attaches to specific attachment sites (viral receptors) 2. Penetration: transfer of viral particle across the cell membrane by endocytosis (in animal and plant viruses), transfer of only the viral genome through the cell membrane (in bacteriophages), fusion of the viral envelope with the host cell membrane (in enveloped viruses) 3. Release of NA from capsid: by enzymes 4. Replication of NA and gene expression: different ds(+/-)DNA (in bacteriophages and animal viruses), DNA is replicated and directly used for transcription and translation ss(+)DNA - first the second strand is formed based on base pairing, dsDNA is then used for synthesis of ssDNA and for gene expression ds(+/-)RNA - one of the strands is used for protein synthesis ss(+)RNA (in bacteriophages, animal and plant viruses) is used directly for protein synthesis, (-)RNA is formed based on base pairing and used for synthesis (+)RNA ss(+)RNA with reverse transcription (in retroviruses) is first reversely transcribed into ss(-)DNA catalysed by viral enzyme reverse transcriptase. Based on base pairing ds(+)DNA is formed and can be incorporated into genome of host cell as a provirus and expressed into proteins. ss(-)RNA (in bacteriophages) - first (+)RNA is formed based on base pairing and then used for protein synthesis and synthesis of (-)RNA. 5. Assembly: viral particles are assembled from proteins and NA 6. Maturation: completed particles are referred to as virions 7. Release: by exocytosis (budding) or by lysis of the host cell ss(+)RNA dsDNA transcription ce mRNA translation replication dsDNA translation ss(-)RNA protein ss(+)RNA ss(+)RNA reversed transcription ss(-)DNA dsDNA protein mRNA virion translation virion ssDNA ss(-)RNA dsDNA transcription ssDNA transcription mRNA translation protein ss(+)RNA translation ss(-)RNA ss(+)RNA protein virion protein virion virion Fig. 30: Scheme of gene expression in different viruses based on type of their nucleic acid. 43 VIROGENIC CYCLE (in animal viruses) or LYSOGENIC CYCLE (in bacteriophages) is characterized by integration of the viral NA into the host cell's genome (integrated genetic material is called a provirus or prophage). It can be transmitted to daughter cells at each subsequent cell division, and its possible subsequent release (induced by stress, e.g. by UV radiation), leads to proliferation of new viruses via the lytic cycle. Bacteriophage Bacterium Lytic cycle Lysogenic cycle Prophage Fig. 31: Lytic and lysogenic cycle in bacteriophage. Examples of viral diseases: Plants: tobacco, tomato or cauliflower mosaic virus disease Animals: foot-and-mouth disease (Aphtae epizooticae) in cloven-hoofed animals (cattle, sheep, goats or pigs), catarrhal fever (or “bluetongue”) (Reoviridae) in ruminants (mainly sheep), avian influenza (Orthomyxoviridae), canine parvovirosis (Parvoviridae), rabies (Rhabdoviridae) in mammals, myxomatosis (Poxviridae) in rabbits. Humans: influenza (Orthomyxoviridae), smallpox or variola (Poxviridae), poliomyelitis called infantile paralysis (Picornaviridae), yellow fever (Flaviviridae, Lat. flavus = yellow), rubella (Togaviridae), ebola haemorrhagic fever (genus Ebolavirus, family Filoviridae), AIDS (Retroviridae), viral hepatitides (hepatitis A - Picornavirus, B - Hepadnavirus, C Flaviviridae), severe acute respiratory syndrome (SARS) (Coronavirus), infectious mononucleosis known as glandular fever (herpesvirus of Epstein-Barr – EBV) and tickborne meningoencephalitis (Flavivirus). Some viruses (e.g. avian influenza virus or rabies virus) can infect both humans and different animal species. Viruses can also cause neoplasia in humans and other species. The main viruses associated with human cancers (oncoviruses) are Papillomavirus (cervical cancer), hepatitis B virus (liver cancer), Epstein-Barr virus (Burkitt's lymphoma, Hodgkin’s lymphoma), human T-lymphotropic virus (adult T-cell leukemia) and Kaposi's sarcoma-associated herpesvirus (Kaposi's sarcoma). Some of the diseases caused by viruses can be prevented by vaccination (e.g. smallpox, canine parvovirosis or rabies, viral hepatitides A and B). VIROIDS are plant pathogens that consist of a small circular ssRNA (a few hundred nucleobases) without the protein coat. Viroid RNA does not code for any protein. The replication mechanism involves RNA polymerase II, an enzyme normally associated with synthesis of mRNA, which instead catalyzes the "rolling circle" synthesis of new viroid RNAs using the viroid's RNA as template. Some viroids are ribozymes (from ribonucleic acid enzyme, also called catalytic RNAs), having catalytic properties which allow self-cleavage and ligation of unit-size genomes from larger replication intermediates. The first viroid (the potato spindle tuber viroid - PSTVd) was identified in 1971. More than 30 different viroids have been identified up to date. 44 SATELLITES are subviral agents composed of nucleic acid (DNA or RNA) that depends on the coinfection of a host cell with a helper virus for their multiplication. When a satellite encodes the coat protein in which its nucleic acid is encapsidated, it is referred to as a satellite virus. Note: Satellite viral particles should not be confused with satellite DNA. VIRUSOIDS belong to satellites. They are infectious subviral particles that infect plants in conjunction with an assistant virus; the assistant virus harbours the virusoid and is required for successful infection. Virusoids consist of a single molecule of circular ssRNA that is several hundred nucleotides long and codes for nothing but its own structure. In size and structure, virusoids are similar to viroids. PRIONS are infectious agents that are composed of only protein. They cause a number of diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, known as "mad cattle disease") in cattle, scrapie in sheep and goats, feline spongiform encephalopathy in cats (FSE) and Creutzfeldt-Jakob disease (CJD), fatal familial insomnia or Kuru in humans. All known prion diseases affect the structure of the brain or other neural tissue, and are currently untreatable. While the incubation period for prion diseases is generally quite long, once symptoms appear the disease progresses rapidly, leading to brain damage and death. Neurodegenerative symptoms can include convulsions, dementia, balance and coordination dysfunction and behavioural or personality changes. Prior to the discovery of prions (in the 1960s), it was thought that all pathogens used nucleic acids to direct their replication. The idea that a protein can replicate without the use of nucleic acids was initially controversial as it contradicts the so-called "central dogma of molecular biology," which describes nucleic acid as the only carrier of information that is able to replicate. There are two forms of prion proteins (PrP): infectious prions (PrPSC – referring to “scrapie”) and endogenous prion proteins (PrPC – from “cellular or common”). Cellular prion proteins with mainly α-helical secondary structure are found on the membranes of nerve cells of healthy people and animals. Infectious prion proteins propagate by converting normal molecules of prion protein into the abnormally infectious isoform by changing their conformation to different folding pattern (with increased β-sheet content replacing normal areas of α-helices). These altered proteins are resistant to proteases, thus accumulating in the cells and forming amyloid plaques that lead to neurodegeneration. ___________________________________________________________________________ TASKS TASK 1: Viruses Draw some of the viruses. Filoviridae (Ebola virus) Rhabdoviridae (rabies virus) Caliciviridae (virus of hepatitis E) Coronaviridae Retroviridae (HIV) Parvoviridae Orthomyxoviridae (influenza virus) Togaviridae (rubella virus) Paramyxoviridae (morbilli virus) Fig. 32: Some examples of viruses (deduction of viral names: filum=fiber, calix=cup, corona=ring, parvus=small, toga=coat). 45 8. Eukaryotes Eukaryotes (domain Eukaryota) include animals, plants, fungi, and protists. According to new classification, there are six kingdoms: Opisthokonta (includes Animalia and Fungi), Amoebozoa, Rhizaria, Excavata, Archaeplastida (includes Plantae, Chlorophyta, Rhodophyta, Glaucophyta) and Chromalveolata (includes Chromista and Alveolata). Eukaryotic organisms can be both unicellular and multicellular. The cells differ in size and shape. They are organised into complex structures (organelles) enclosed within membranes. E.g. nucleus, mitochondria and chloroplasts are organelles with two membranes or endoplasmic reticulum and Golgi apparatus are organelles with one membrane. The average size of an eukaryotic cell is 10-100 µm. The presence of a nucleus gives these organisms their name (from Greek eu means “good or true” and karyon means “nut”). Eukaryotic cells are surrounded by selectively permeable plasma membrane. Moreover, plant and fungal cells have a cell wall, a rigid layer outside the membrane, providing the cell with structural support and protection. Eukaryotic ribosomes have sedimentation coefficient 80S, each consisting of a small (40S) and large (60S) subunit. Eukaryotic somatic cells reproduce by mitosis, where one cell divides to produce two genetically identical cells. Gametes (sexual cells) are the result of meiosis, when one diploid cell is divided into four haploid cells. NUCLEUS is surrounded by a double membrane (nuclear envelope) with pores important for transport processes. Chromosomes are located in the nucleus. They are formed by chromatin (consisting of linear DNA molecules and proteins, mainly histones). The structure (level of condensation or packaging) of chromatin varies significantly between different stages of the cell cycle (euchromatin and heterochromatin). DNA contains many genes composed of introns (noncoding part) and exons (coding part), regulatory elements and also various non-coding nucleotide sequences. Chromosomes vary between different organisms and their number and structure (called karyotype) is species specific (see Chapter 15.1.). Nuclei of diverse cells differ in size and shape. Some highly specialized cells, such as erythrocytes (red blood cells) in mammals, lack nuclei. NUCLEOLUS is a non-membrane structure found within the nucleus. It is formed around specific genetic loci called Nucleolar Organising Regions (NOR's), that are composed of tandem repeats of rRNA genes and which can be found in several different chromosomes. Nucleolus provides ribosome biogenesis, including rRNA synthesis by transcription, and formation of the 40S and 60S subunits of ribosomes, that are subsequently exported through the nuclear pores to the cytoplasm where they remain free or will become associated with the endoplasmic reticulum. The other nucleic acid tRNA is also synthesized in nucleolus. Size and number of nucleoli can differ in nuclei of individual cells, especially according to their metabolic activity. Nucleoli are present only in interphase cells. ENDOPLASMIC RETICULUM (ER) is an organelle that forms interconnected network of tubules, vesicles, and cisternae held together by the cytoskeleton. There are three types of ER. Rough endoplasmic reticulum with ribosomes on its surface is responsible for protein synthesis and their transport to be used in the cell membrane (e.g. transmembrane receptors and other integral membrane proteins), or to be secreted (exocytosed) from the cell (e.g. digestive enzymes). Smooth ER without ribosomes on its surface has functions in several metabolic processes, such as synthesis and storage of lipids, steroids, glycogen, and other macromolecules, metabolism of carbohydrates, regulation of calcium concentration. Sarcoplasmic reticulum is a special type of smooth ER found in smooth and striated muscles. It contains large stores of calcium, which it sequestered and then is released when the muscle cell is stimulated. 46 GOLGI APPARATUS (GA) or GOLGI COMPLEX is organelle with a single lipid bilayer membrane found in most eukaryotic cells. It is composed of flattened disks known as cisternae that are separated and communicate by membrane-bound vesicles. Its main function is chemical modification (glycosylation, phosphorylation, sulfatation etc.) of macromolecules synthesized by ER and packaging them for cell secretion (exocytosis) or for use within the cell. It is involved in the creation of lysosomes and plays an important role in the synthesis of proteoglycans (molecules present in the extracellular matrix of animals) and carbohydrate synthesis. The Golgi complex in plant cells is called dictyosome. Ríbosomes Vesicles Nucleolus omplex Pore Nucleus complex Rough ER Golgi complex Fig. 33: Secretion pathway of proteins. MITOCHONDRION is a double membrane organelle 1-10 µm in size. The number of mitochondria in a cell varies widely according to organism and tissue type (from one to thousands). The most important role of mitochondrion is cell respiration when chemical energy is released from organic matters and oxidative phosphorylation when energy is stored in macroergic bonds of ATP (adenosine triphosphate). Glucose is cleaved into two pyruvate molecules by anaerobic glycolysis in cytoplasm of the cell to form two ATP and two NADH molecules. Pyruvate is actively transported across the mitochondrial membranes into the matrix where it is oxidized and combined with coenzyme A to form acetyl-CoA, CO2 and two NADH molecules. Acetyl-CoA enters the citric acid cycle (Krebs cycle) and is oxidized to CO2. Molecule of GTP (readily converted to ATP) and reduced cofactors (three NADH and one FADH2) are also produced. Redox energy from NADH and FADH2 is transferred to oxygen in several steps via the electron transport chain in the inner membrane of mitochondrion. Protein complexes (NADH dehydrogenase, cytochrome c reductase and cytochrome c oxidase) located in the inner membrane, are used to pump hydrogene protons into the intermembrane space. As the proton concentration increases in the intermembrane space, a strong electrochemical gradient is established and protons can return to the matrix through the ATP synthase complex and their potential energy is used to synthesize ATP from ADP and inorganic phosphate. Mitochondria have their own independent genomes (circular DNA with high proportion of coding DNA) and ribosomes similar to bacterial ones. However, most of the proteins necessary for mitochondrial functions are encoded by genes in the cell nucleus. Mitochondria divide by binary fission that is similar to bacterial cell division. According to the endosymbiotic theory, mitochondria are descended from ancient bacteria, which were engulfed by the ancestors of eukaryotic cells more than a billion years ago. Matrix (inner space) Mitochondrion Intermembrane space Inner membrane forming cristae Outer membrane 47 PLASTIDS are organelles found in plants and eukaryotic algae. Plastids are the site of production and storage of important chemical compounds used by the cell. Plastids often contain pigments determining the cell's color (green chloroplasts containing chlorophyll or chromoplasts for synthesis and storage of other pigments). Plastids are also important for other functions e.g. leucoplasts for monoterpene synthesis, amyloplasts for starch storage, elaioplasts for storing fat and proteinoplasts for storing and modifying proteins. CHLOROPLASTS are oval double membranous organelles. Chloroplasts absorb light and use it in conjunction with water and carbon dioxide to produce sugars (glucose) and oxygen in process called photosynthesis (see equation below). Chloroplasts capture light energy to conserve free energy in the form of ATP in process of photosyntetic phosphorylation (on the membrane of tylakoids) and reduce NADP to NADPH. sunlight H2O + CO2 C6H12O6 + O2 + energy Chloroplasts are generally considered to originate from endosymbiotic prokaryotes (cyanobacteria). Chloroplasts have their own genomes (circular DNA molecules). Tylakoids forming grana Chloroplast Stroma (inner space) Inner and outer membrane PEROXISOMES are organelles with a single lipid bilayer membrane. Their major function is the breakdown of fatty acid molecules in a process called beta-oxidation. In liver or kidney cells, the peroxisomes detoxify various toxic substances that enter the blood (e.g. ethanol). They also contain oxidative enzymes to destroy toxic peroxides. Like lysosomes, peroxisomes are part of the secretory pathway of a cell. LYSOSOMES are small vesicular organelles that contain digestive enzymes (about 40 different acid hydrolases) that digest food particles, engulfed viruses or bacteria or old organelles. They are created by addition of hydrolytic enzymes to endosomes from the Golgi apparatus. The interior of the lysosomes is more acidic than the cytosol (pH < 5). This is enabled by a single membrane surrounding the lysosome that stabilizes the low pH by pumping in protons (H+) from the cytosol via proton pumps and chloride ion channels. The membrane also protects the rest of the cell from enzymes within the lysosome. VACUOLES are present mainly in plant cells. They are surrounded by a membrane called the tonoplast. Vacuoles contain large amounts of a liquid composed of water, enzymes, inorganic ions, stored saccharides, pigments and other substances including toxic products removed from the cytosol to avoid interference with metabolism. The other role of the central vacuole is to maintain turgor pressure against the cell wall. Due to osmosis, water will diffuse into the vacuole, placing pressure on the cell wall. Unicellular eukaryotes (e.g. protists) can have contractile vacuoles involved in osmoregulation by removing excess water out of a cell and digestive vacuoles containing enzymes for the digestion. INCLUSIONS are aggregates of chemical substances presented in cell cytoplasm: a) Storage – starch grains in plant cells, glycogen granules in the liver and muscle cells and lipid droplets in fat cells. b) Crystalline – crystals of various types, e.g. calcium oxalate crystals of various shapes (raphids, druses or styloids), that are used to dispose of excess calcium. c) Pigment – pigment granules in certain cells of skin and hair. Pigment containing cells are called chromatophores and can be grouped into subclasses based on their color 48 (e.g. black/brown melanophores, yellow xanthophores, red erythrophores, white leucophores or blue cyanophores). CYTOSKELETON is composed of various proteins both filamentous and globular. It is a dynamic system that maintains cell shape, enables cellular motion, plays important roles in both intracellular transport and cellular division. Eukaryotic cells contain three main kinds of cytoskeletal filaments: a) Intermediate filaments (8 - 12 nm in diameter) are composed of different proteins, such as vimentins, keratin (found in skin cells, hair and nails) or lamin (structural support to the nuclear envelope). Their function is to maintain cell-shape, organise the internal 3D structure of the cell, anchor organelles, form the nuclear lamina and sarcomeres and participate in some cell-cell and cell-matrix junctions. b) Microtubules (25 nm in diameter), comprise 13 protofilaments which are polymers of globular protein called tubulin. They are organised by centrosome. Microtubules play important role in intracellular transport, movement (cilia and flagella), cell division (mitotic spindle) or synthesis of the cell wall in plants. c) Microfilaments (actin filaments) (7 nm in diameter) are composed of two chains of globular protein actin. Microfilaments are concentrated under the cell membrane, to help maintain cellular shape. They form cytoplasmic protuberances (pseudopodia or microvilli), they are important for amoeboid movement, cytokinesis in animal cells (cleavage and furrow formation) and for muscular contraction (along with myosin). 8.1. Plant cell Plant cells contain chloroplasts (and other plastids) and vacuoles with cell fluid (containing stored saccharides, pigments, etc.) and are surrounded by cell wall composed of cellulose. They often contain storage or crystalline inclusions. ANTHOCYANINES belong to the plant pigments present in the vacuoles of some species and some cellular types. Anthocyanines are present in various fruits e.g. raspberry, cherry, red wine, blackcurrant or wild privet berries (Ligustrum vulgare) and flowers e.g. petals of violet or lungwort (Pulmonaria), in leaves (red cabbage), seed coats (black soybean), stems (begonia) or roots (red beet). They change their color according to pH from red (in acidic pH), through purple (in neutral pH), to blue (in alkaline pH). They contribute to autumn leaf coloration together with orange carotenoids and yellow xanthophylls. Anthocyanines protect cells from high-light damage by absorbing bluegreen and UV light, enhance frost hardiness and limit water loss during dry spells. ___________________________________________________________________________ TASKS TASK 1: Chloroplasts NP: leaf of aquatic plant Vallisneria sp. or leaf of moss (Brachythecium rutabulum) Prepare NP from leaf and observe plant cells with cell wall and round green chloroplasts. . Chloroplasts Cell wall Fig. 34: Plant cells with cell wall and chloroplasts. 49 TASK 2: Vacuoles NP: berries of Wild Privet (Ligustrum vulgare) Cut the berry and impress it on the slide glass, cover with cover glass and observe the violet coloured vacuoles and green chloroplasts. Then add a drop of NaOH (alkaline solution) to the one edge of glass and drop of acetic acid (acidic solution) to the second one. How does the color of vacuoles change according to different pH? TASK 3: Crystalline inclusions of calcium oxalate NP and PP: the outer peel (epidermis) of onion (Allium cepa) – “cibule” and stalks of Begonia and Pothos Observe styloids (prismatic crystals) in outer peel of onion, druses in fluid from Begonia stalk and raphids (needle-like crystals) in fluid from Pothos stalk. TASK 4: Pollen grains PP: the pollen grains - “pylová zrna, pyl” Observe the various shapes of pollen grains from different plant species. Pollen grains vary in contrast; it is difficult to find them, and it is helpful to adjust the light to lower intensity. Chloroplasts Druses Vacuole Styloids Raphids A B C Fig. 35: A – vacuoles and chloroplast in berries of Ligustrum vulgare, B – crystalline inclusions of calcium oxalate, C – pollen grains. 8.2. Animal and protozoan cells Animal and protozoan cells lack cell walls. Some of them can have structures such as flagella, cilia or pseudopodia (depending on species and tissue type). Plastids and vacuoles (contractile and/or digestive) are present only in some protozoans. ___________________________________________________________________________ TASKS TASK 1: Epithelial cells of human buccal mucosa Take a sample from your mouth cavity (inner side of cheek) using cotton swab. Then impress the swab against a slide, add a drop of water and cover the glass. Find and observe scattered transparent polygonal epithelial cells containing nucleus (start with a small magnification, low brightness, use meandering search, locate the correct optical plane, and beware of artefacts!). 50 TASK 2: Nerve cells (neurons) PP: the spinal cord of pig (Sus) or rat - histologic cross section, stained with haematoxylin-eosin - “mícha” Find the neurons in the so-called grey matter in the ventral spinal horns. Observe the large (particularly in the pig), intensively pink-stained neurons with nuclei and projections (dendrits). Cytoplasmic membrane Dendrit Nucleus Nucleus Cytoplasmic membrane B A Fig. 36: A – epithelial cells of human buccal mucosa, B – nerve cell. TASK 3: Shape and number of nuclei PP: leucocytes - “bílé krvinky”, protozoan Spirostomum - “korálkovité jádro/růžencovité jádro”, protozoan Paramecium - “mikronukleus a makronukleus”, protozoan Opalina ranarum - “mnohojaderná buňka” Using small magnification, find a place with a higher density of leucocytes (violet dots scattered among more numerous pink erythrocytes) and observe the shapes of the nuclei: round in lymphocytes, kidney- or bean-like in monocytes, lobular, segmented or polymorphic in mature forms of granulocytes, s-shaped and band shaped in younger forms. Using a meandering movement, find a pink-stained ciliate protozoan Spirostomum with a beadshaped nucleus. Observe violet coloured macronucleus (large nucleus, vegetative nucleus) and micronucleus (small nucleus, generative nucleus) in ciliate protozoan Paramecium. Observe the numerous purple-stained nuclei within the greenish cytoplasm of protozoan Opalina ranarum living as a commensal in the cloaca of frogs. Round nucleus Bean-like shaped nucleus Cell without nucleus (erythrocyte) Lobular, S-shaped and horseshoe -shaped nucleus Macronucleus Polynuclear cell Micronucleus Bead-shaped nucleus Fig. 37: Different shapes and numbers of nuclei. 51 TASK 4: Cell organelles - Golgi apparatus, mitochondria PP: Golgi apparatus is in the silver stained cross section of a rat intestine - “Golgiho aparát”, liver selectively stained for mitochondria after Heidenhain - “mitochondrie” Find an intact villus projecting into the lumen (interior) of the intestine. Find the layer of cylindric epithelial cells (enterocytes) on its surface. Note the violet elongated nuclei in the basal part of enterocytes and the Golgi apparatus in the apical (peripheral, luminal) part of enterocytes (above the nuclei). It is a round-shaped cluster of brown-black dots (silver stain) or is pale-colored. Observe occasional cells with two nuclei in the liver and mitochondria as tiny blue-black points (like powder) inside the liver cells (hepatocytes). Golgi apparatus Nucleus Villus A B Fig. 38: A – cross section of intestine with villus, B – enterocyte with Golgi apparatus (GA) and nucleus. nucleus with nucleolus mitochondria Fig. 39: Mitochondria in liver cells (stained after Heidenhain). TASK 5: Pigment inclusions PP: melanophores or melanin containing cells in frog skin - “melanofory” Observe the melanin pigment granules inside the cells (melanophores) with numerous branching projections. Melanophores or, more generally, chromatophores (Greek phorein = to carry), are responsible for coloration in animals (e.g. frogs) and sometimes enable color changes (in animals such as chameleons). Nucleus Melanin Fig. 40: Melanophores of frog containing pigment melanin. 52 9. Research methods in biology 9.1. Cell and tissue cultures Isolated cells (derived from multicellular eukaryotes), tissues and even organs or their parts can be grown and maintained in laboratory conditions, i.e. in vitro (from Latin „within the glass“) or ex vivo („out of the living“) in contrast to their normal occurrence in vivo („within the living“). Cell and tissue cultures are nowadays important for many biomedical and biotechnology applications and for basic research. They represent important tool for manufacturing of viral vaccines and other biotechnology products (such as monoclonal antibodies or enzymes and synthetic hormones produced by recombinant DNA technology). Cell cultures are essential for cultivation of intracellular parasites (e.g. viruses or protozoans, such as Plasmodium), in controlling diseases and in study of the reaction to certain agents. They can be used also in toxicity tests and in the development and testing of new drugs. In some applications, well-established cell culture models can reduce animal experiments. HISTORY S. RINGER, the 19th-century English physiologist, developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body. W. ROUX removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days. Thus he established the principle of tissue culture in 1885. The problem was a long-term cultivation because of bacterial contamination. H. EAGLE was an American physician and pathologist, who was the first to define exact composition of a culture medium (Eagle's minimal essential medium). R. G. HARRISON established the methodology of tissue cultures in the beginning of 20 th century. He demonstrated the growth of frog nerve cell processes in a medium of clotted lymph. P. ROUS and F.S. JONES used proteolytic enzyme trypsin to digest tissue for separation of individual cells. They demonstrated repeated passages of the cells. L. HAYFLICK and P. MOORHEAD found out that human fibroblasts cultivated in tissue cultures die after several generations (see phase growth curve of the tissue culture). M. WIGLER and R. AXEL developed method for injecting mammal genes into the cells. J. F. ENDERS, T. H. WELLER and F. C. ROBBINS carried out cell culture research with application in virology in the 1940s and 1950s that allowed preparation of purified viruses for the manufacture of polio vaccine. They were awarded a Nobel Prize for their discovery of a method of growing the virus in monkey kidney cell cultures. TYPES OF CULTURES Tissue culture is the growth of tissues separated from the organism. Tissue culture commonly refers to the culture of animal tissues, while the more specific term plant tissue culture is used for plants. The advantage of these cultures is that cells in tissue can interact with each other similarly as in organism. Cell culture is the process by which prokaryotic or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. Both adherent cells (e.g. epithelial cells that require a surface, to which they attach by extracellular matrix proteins, such as collagen) and floating (or suspension) cells (especially cells that naturally live in suspension, such as lymphocytes) can be cultured ex vivo. Cells that are cultured directly from an organism are known as primary cell cultures. 53 With the exception of some tumour cells, most primary cell cultures have a limited lifespan. After a certain number of population doublings cells undergo the process of senescence and stop dividing. An established cell line (or immortalised cell line) has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. There are numerous well established cell lines representative of particular cell types. Examples of cell lines: Cell lines Kc167 MDBK MDCK Vero HeLa BHK21 SP2 Cell type Embryonal Epithelial Epithelial Epithelial Epithelial Fibroblast Plasma cells Origin Fruit fly Cattle Dog Vervet Monkey Human Hamster Mouse Way of cultivation Adherent Adherent Adherent Adherent Suspension Suspension Suspension (Note: HeLa line - cancer cells from Henrietta Lacks, who died at 31 from uterine cervical carcinoma). CULTIVATION VESSELS (bottles or flasks) can be made of glass or plastic (named after Roux). In vessels, cells can grow in suspension (e.g. blood cells) or as adherent cultures covered by liquid culture medium. Cells growing in cultivation vessels can be observed by inversion microscope. GROWTH MEDIUM (or culture medium) used for animal cell or tissue cultures is usually liquid (compared to gel media mostly used to culture bacteria). Growth medium has to have defined pH (optimum 7.4), concentration of ions and has to contain all necessary nutrients (carbohydrates, amino acids, vitamins, fatty acids, etc.). Phenol red is added to medium as an indicator of pH. If red color of indicator is getting to be yellow (nutrients are consumed, metabolites accumulate and pH decreases), it is the signal for exchanging medium with fresh one. Eukaryotic cells derived from whole organisms and grown in culture usually cannot grow without the addition of, for instance, hormones or growth factors which usually occur in vivo, which are usually supplemented by addition of blood serum to the medium. Antibiotics can also be added to the growth media to prevent bacterial contamination. CULTURING AND PASSAGING Cells have to be cultured in well-defined conditions (typically, 37°C, 5 % CO2 for mammalian cells) in a thermostat. Among the common manipulations carried out on culture cells are media changes and passaging cells (also called “splitting cells”), that involves transferring a small number of cells into a new vessel after they reach certain culture density. All these manipulations require a sterile technique that aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety box (often with laminar air flow) to exclude contaminating microorganisms. Antibiotics can also be added to the growth media. The growth of the cells is characterized with a cell growth curve (see Figure below). Cells grown in monolayer proliferate to a confluent state in which the cells cover the growth surface of the flask. Cellto-cell contact can stimulate cell cycle arrest, causing cells to stop dividing known as contact inhibition. Some cells can be maintained in this phase of growth for days to weeks, while others require trypsination and subculture to survive. Cells can be cultured for a longer time if they are passaged regularly, as it avoids the cell aging associated with prolonged high cell density. Cells can be grown in suspension or adherent cultures. 1) Adherent cells grow attached to the surface of cultivation vessels by proteins of extracellular matrix e.g. laminin, fibronektin and collagen. Most cells derived from solid 54 tissues are adherent. During cultivation, the old medium can be removed directly by aspiration and replaced with fresh one. For passaging adherent cultures, cells first need to be detached with trypsin. A small number of detached cells can then be used to seed a new culture. 2) Floating (or suspension) cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are cell lines that have been modified to be able to survive in suspension cultures so they can be grown to a higher density than adherent conditions would allow. Before medium exchanging or cell passaging, the cells should be divided from the medium by centrifugation. Suspension cultures are then easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. III cell number IV II I I I – Lag phase (growth stagnation) II – Logarithmic phase III – Stationary phase (peak) IV – Decrease, possibly death A B Time Fig. 41: A – plastic bottles for cell cultivation, B – phase growth curve of a tissue culture __________________________________________________________________________________________ TASKS TASK 1: Tissue culture PP: stained kidney tissue culture (monolayer) Observe and draw rabbit kidney tissue cells. Cubic cell in anaphase Spindle-shaped cell in interphase Cubic cell in prophase Spindle-shaped cell in metaphase Fig. 42: Cells from tissue culture in different phases of mitosis or in interphase. 55 TASK 2: Work with inversion microscope NP: adherent cells from cell culture Observe adherent cells in cell culture growing in monolayer. Then pour off the medium from the bottle and rinse the cells with phosphate buffered saline and add 2 ml of trypsin. After the trypsinization you can observe loose cells, sometimes in clusters. Gentle shaking the bottle or gentle heating can help to release the cells from the bottom of the cultivation vessel. Add the medium with serum that inhibits the effect of trypsin to protect the cells. Transfer cells floating in the medium to a beaker and observe them under a light inversion microscope. What is the shape of cells now? 9.2. Molecular biology techniques Molecular biology (MB) is a scientific discipline that deals with study of biology at a molecular level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the structures of macromolecules (DNA, RNA, proteins, etc.) and the interactions between them and learning how these interactions are regulated and how they are related to processes in the living systems. Researchers in molecular biology use specific techniques native to molecular biology, but combine these with genetics and biochemistry approaches. Molecular biology methods are used both in basic genetic research or genetic engineering and in applied disciplines, such as medicine, clinical diagnostics, criminalistics, archaeology or forensic medicine. Techniques of molecular biology include e.g. physical and chemical separation techniques, such as chromatography or electrophoresis, but also recombinant molecular techniques (e.g. cloning), the use of arrays or hybridization techniques, such as various types of blotting. The most important techniques are purification and separation of nucleic acids, their amplification, methods of NA manipulation, DNA sequencing and various methods of gene expression analyses (studies of transcription and translation). Principles of the most widely used techniques are explained below, including the examples of their possible usage. DNA ISOLATION is the first step of most of the MB techniques. DNA can be isolated from various types of materials, e.g. all nucleated cells, prokaryotic cells, viral particles, tissues. DNA isolation comprises three basic steps: 1) Mechanical or chemical disruption of cells (or tissues, virions, etc.) using enzymes (lysozyme, cellulase) and/or detergents (such as sodium dodecyl sulphate), to remove membrane lipids. 2) Removing contaminants (proteins, RNA, or lipids) using mainly enzymes (proteinases) to remove cellular and histone proteins bound to DNA or RNA. 3) DNA extraction by precipitating the DNA with an alcohol (ethanol or isopropanol). Since DNA is insoluble in these alcohols, it will aggregate together giving a pellet on centrifugation. Phenol-chloroform extraction is a commonly used technique for DNA isolation. This method relies on phase separation by mixing the aqueous sample with a solution containing phenol and chloroform and on centrifugation, resulting in an upper aqueous phase containing dissolved DNA, lower organic phase formed by phenol containing proteins and interphase containing denatured proteins and cell debris. Subsequently, DNA is recovered from the solution by precipitation with ethanol and by centrifugation. DNA CLONING in general describes a procedure of isolating a defined DNA sequence and obtaining multiple copies of it. DNA can be cloned into a vector (e.g. plasmid or a viral vector), that is capable of replicating in a host cell. Restriction enzymes are used for introduction of target sequence into vector. The purpose of cloning DNA into vectors is 56 oocyte (egg cell) - Sample Sample-mixture of DNA fragments of different size Standard Standard usually to isolate, multiply, or express the insert in the target cell. Expression cloning is used to study protein functions. In this technique, DNA coding for a protein of interest is cloned into a plasmid that can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation (via uptake of naked DNA), conjugation (via cell-cell contact) or by transduction (via viral vector). Introducing DNA into eukaryotic cells (e.g. by electroporation or microinjection) is called transfection. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection. GEL ELECTROPHORESIS belongs to the most important tools for NA and protein separation for analytical purposes, but it may be used also as a preparative technique prior to use of other methods such as RFLP, DNA sequencing or Southern blotting for further characterization. The basic principle is that DNA, RNA, and proteins can be separated by means of electric current applied to a gel matrix. DNA and RNA can be separated on the basis of size by running through an agarose gel. Proteins can be separated on the basis of size by using an SDS-PAGE gel or on the basis of size and their electric charge by using what is known as a 2D gel electrophoresis. In DNA gel electrophoresis, negatively charged DNA molecules (the main source of negative charge are the phosphate groups) migrate in the electric field towards an anode. The separation gel is usually a crosslinked polymer (acrylamide or agarose). Molecules move through the matrix at different rates that are approximately inversely proportional to their size (smaller fragments move faster, while longer molecules move slower). After the electrophoresis is complete, the separated fragments in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for visualisation of distinct bands. Ethidium bromide (EtBr) is an intercalating agent that fluoresces when exposed to ultraviolet light (intensifying almost 20-fold after binding to DNA). It is used to visualise NAs in combination with an UV transilluminator (a device that projects UV radiation). EtBr should be treated carefully, because it is mutagenic and carcinogenic. Molecular weight size markers (or “ladders”) (mixtures of molecules of known sizes) are used to determine the size of fragment(s) in unknown sample. Separated molecules can be detected also by radioactive staining or by hybridization with a labelled probe (short oligonucleotide that binds on the basis of complementarity to a target sequence). Separated NA molecules can be isolated from the gel for further applications. Long fragments Source Short fragments gel Fig. 43: Gel electrophoresis. 57 PCR (polymerase chain reaction) is a method of in vitro enzymatic replication using DNA polymerase to amplify certain DNA sequence. It was developed in 1984 by Kary Mullis, who was awarded the Nobel Prize in Chemistry in 1993 for his work on PCR. It is a chain reaction, i.e. DNA template is exponentially amplified in series of steps that are periodically repeated. With PCR it is possible to amplify a single or few copies of DNA sequence, generating millions or more copies of the DNA sequence (generally 2n-1 copies, where n is number of cycles). PCR usually employs a thermostable DNA polymerase (e.g. Taq polymerase, an enzyme originally isolated from thermophilic bacterium Thermus aquaticus). This DNA polymerase enzymatically assembles a new DNA strand from DNA nucleotides (based on their complementarity) by using ssDNA as a template and DNA oligonucleotides (primers), which are required for initiation of DNA synthesis. Thermal cycling (heating and cooling the PCR sample to a defined series of temperature steps) is necessary to physically separate (denaturate) the two strands of a DNA double helix (at high temperature), to be subsequently used as templates during the DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The amount of the DNA target is doubled after each cycle, leading to exponential (geometric) amplification of the specific DNA fragment. The selectivity of PCR results from the use of a pair of primers (chemically synthesized oligonucleotides) that are complementary to the DNA region targeted for amplification (single gene, a part of a gene, or a non-coding sequence). Each of the two primers binds to a different DNA chain with their 3'-OH-ends in opposite directions. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs. The PCR is commonly carried out in a reaction volume of 10200 μl in small reaction tubes in a thermal cycler (thermocycler), a laboratory apparatus capable of heating and cooling the reaction tubes to achieve the temperatures required at each step of the reaction (see below). PCR usually consists of 20 to 40 cycles; each typically consists of three discrete temperature steps. The cycling is often preceded by a single temperature step at a high temperature (>90°C). The temperature and the length of each step depend on a variety of parameters, including the enzyme used for the DNA synthesis, and the melting temperature (Tm) of the primers. A basic PCR reaction requires several components and reagents: Template DNA that contains the DNA region to be amplified. Pair of primers, which are complementary to the DNA regions at the 5' (five prime) or 3' (three prime) ends of the DNA region. dNTPs (deoxyribonucleotide triphosphates: dATP, dCTP, dGTP and dTTP) Thermostable DNA polymerase Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Bivalent cations (usually Mg2+) and monovalent cation potassium ions. Each cycle of PCR usualy consists of 3 steps: 1) Denaturation (typically 95°C for 20-30 seconds), that causes melting of the DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding ss DNA. 2) Annealing of primers (50-65°C for 30-90 seconds) to the ss DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. The polymerase binds to the primer-template hybrid and begins the DNA synthesis. 3) Extension (or elongation) (typically 72°C for 1 or 2 minutes) of growing DNA chain catalyzed by DNA-polymerase, that synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction. The extension time depends on the length of the DNA fragment to be 58 amplified (DNA polymerases are able to polymerize approximately thousand bases (1 kb) per minute). An example of PCR procedure: Preheating (95°C /2-10 min.) 1. Denaturation (95°C/20-45 s.) 2. Annealing of primers (50–65 °C/30-90 s.) 3. Extension (72°C/60-120 s.) Final extension (72°C/5 min.) These steps cyclically repeat (20-40 x) Final PCR product (amplified DNA sequence) can be further analyzed e.g. by gel electrophoresis to determine the size of the product, by enzymatic cleavage to examine the restriction fragment spectra (e.g. by RFLP – see below), or by hybridization with a labelled probe complementary to part of the sequence. A common variation of PCR technique is reverse transcription PCR that is used to amplify a sequence of the RNA molecule (usually the mRNA) and is preceded by a reaction using the reverse transcriptase to convert RNA to so-called cDNA (complementary DNA). Asymmetric PCR is used to amplify only one strand of the original DNA by using only one primer, it is used for DNA sequencing (see below). The usage of PCR: PCR is used for variety of applications in both basic and applied research e.g. for DNA sequencing, isolation of genes and their parts, for preparation of labelled probes and for determination of mutations or gene polymorphisms. Reverse transcription PCR is widely used to determine gene expression. PCR is also important for many clinical disciplines (in prenatal diagnostics for diagnosis of hereditary diseases or for sex determination, for detection of pathogenic microorganisms or oncogenes), in zoology (DNA-based phylogeny), archeology, criminalistics (genetic fingerprints used for identification of individuals) or in forensic medicine (e.g. for paternity testing). NUCLEIC ACID HYBRIDIZATION represents pairing of single-stranded NAs based on complementarity between nucleotides of two DNA or RNA molecules (or DNA and RNA). In fact, it is an opposite process to denaturation of double stranded molecules. The NAs molecules used for hybridization may come from different organisms, or can be artificially synthesized. NAs are usually hybridized with a labelled probe (short oligonucleotide with known sequence) used to detect complementary target sequence in DNA or RNA molecule. Probes can be labelled radioactively or by a fluorescent dye. Hybridization with a labelled probe can be used for target sequence detection in intact cells (in situ hybridization). There are two basic applications based on nucleic acid hybridization: Southern Blotting (DNA hybridization) and northern blotting (RNA hybridization). Southern blotting was developed by E.M. Southern. In this method, DNA is digested with a restriction enzyme and the fragments are separated by gel electrophoresis. The gel is then overlaid with a filter (made of nylon or nitrocellulose), to which the DNA fragments are transferred (this process is called blotting). The fragments on the filter have the same positions they had on the electrophoretic gel. Subsequently the filter is incubated with a labelled probe, which hybridizes to the complementary DNA sequence, thus detecting it. RNA sequences are identified by northern blotting: RNA is extracted and separated according to size by electrophoresis. The rest of the procedure is similar to Southern Blotting. Northern blotting is used to study gene expression (e.g. whether specific mRNA is present in different cell types). Procedure of Southern blotting and hybridization: 1) Synthesis and labelling of the probe 2) Enzymatic cleavage of DNA and fragment separation by gel electrophoresis 59 3) DNA denaturation and transfer of ssDNA molecules to a filter (blotting) 4) Incubation of the filter with the labelled single stranded probe → hybridization of the probe to the complementary DNA sequence immobilized on the filter 5) Rinsing the filter to remove the unbound probe 6) Detection of the bound probe - visualization by autoradiography in case of radioactive probe or fluorescence determination Labelled ss probe Denaturation Hybridization ss DNA dsDN A Labelled ss probe binds to complementary sequence of ss DNA Fig. 44: DNA hybridization. RFLP (restriction fragment length polymorphism) is method based on enzymatic cleavage of DNA molecules in specific sequence. Different restriction endonucleases cleave DNA molecules in specific restriction sites based on DNA sequence. Obtained DNA fragments can be separated by gel electrophoresis and detected polymorphism in restriction fragment numbers or lengths will give us the information about differences in DNA sequences. These polymorphisms can be caused by insertions, deletions or substitutions of bases that change the sequence of the restriction site. offspring mother father ? father? offspring mother father father? Electrophoresis Fig. 45: Principle of RFLP – paternity test (DNA from mother, offspring and fathers is cleaved by the same enzyme to form fragments that are separated by gel electrophoresis and one father is excluded based on compairing fragments). DNA SEQUENCING is a method that is used to determine the primary DNA structure, i.e. the sequence of nucleotides in DNA. Sequencing is based on preparation and detection of DNA fragments that differ in size in one nucleotide from each other. The most commonly used method is chain-termination method developed by Frederick Sanger in 1975. The Sanger method is based on DNA amplification by PCR with the use of dideoxynucleosides triphosphates (ddNTPs) as DNA chain terminators. These ddNTPs lack a 3'-OH group required for the formation of a phosphodiester bond between two 60 nucleotides during DNA strand elongation. Their incorporation into the nascent (elongating) DNA strand therefore terminates DNA strand extension, resulting in synthesis of DNA fragments of varying length. The DNA sample is divided into four separate sequencing reactions, each of them containing different ddNTP (ddATP, ddGTP, ddCTP, or ddTTP), in addition to all four types of the standard dNTPs, the DNA template, primer, and the DNA polymerase. DNA is amplified using asymmetric PCR, i.e. only one strand of the original DNA is amplified due to use of only one primer. The newly synthesized DNA fragments are separated according to size by gel electrophoresis. Each of the four reaction mixtures contains: DNA template one primer four types of normal deoxynucleotides (dATP, dGTP, dCTP and dTTP) Dideoxynucleotide (ddNTP: ddATP, ddGTP, ddCTP, or ddTTP) – small amount, different in each reaction mixture DNA polymerase HO OH HO H H H Fig. 46: Chemical structure of dideoxynucleoside triphosphate and the principle of synthesis termination after incorporation of ddNTP into nascent DNA strand. Procedure of Sanger DNA sequencing: 1. Obtaining the DNA to be sequenced by extraction and subsequent amplification by PCR 2. Division of the sample into four reaction tubes, each containing different ddNTP (ddCTP, ddGTP, ddATP, or ddTTP). 3. Asymmetric PCR, during which ddNTP terminators are incorporated to appropriate positions of nascent DNA molecule, resulting in each of the four samples containing fragments terminated by one type of ddNTP. 4. Denaturation of synthesised DNA. 5. Electrophoretic separation of fragments with a resolution of just one nucleotide. 6. Fragment detection. 61 Nowadays, automated DNA sequencing using labelled terminators is the most commonly used method for DNA sequencing. In this dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with a different fluorescent dye, each fluorescing at a different wavelength. DNA amplification using this method can be performed in a single reaction. Obtained fragments are separated by capillary electrophoresis and detection and recording of dye fluorescence are performed using automated computer-controlled sequence analyzers (DNA sequencers), resulting in data output as fluorescent peak trace chromatograms with peaks of different colors representing the nucleotide sequence. This method is very fast and easy to perform and offers high throughput. Fig. 47: Principle of DNA sequencing using Sanger method. ARRAYS enable to analyze large quantity of different DNA (or RNA) sequences. Microarrays and macroarrays are collection of spots attached to a solid support (e.g. microscope slide), where each spot contains one or more single-stranded oligonucleotide fragment complementary to a single DNA sequence (similar to Southern blotting). A variation of this technique allows comparison of gene expression of an organism at a particular stage in development, or of two different tissues, such as a healthy and cancerous tissue (expression profiling). In this technique the RNA in a tissue is isolated and converted to labelled cDNA. This cDNA is then hybridized to the fragments on the array and visualized. METHODS FOR PROTEIN ANALYSIS. Proteins can be separated according to their size using gel electrophoresis, usually sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS is an anionic detergent which denatures secondary and non-disulfide-linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Proteins are then transferred from the gel to a membrane (nitrocellulose, nylon, etc.) by technique called western blotting. Target proteins bound to the membrane can be detected by antibodies (labelled with enzymes that digest chemiluminescent substrate), that specifically bind to the protein and can be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, or sheep (polyclonal antibodies) or produced in cell culture (monoclonal antibodies). Two-dimensional gel electrophoresis (2-D electrophoresis) is commonly used to analyze proteins. This method enables separation of proteins according to their isoelectric point (the pH at which a molecule carries 62 no electrical charge) in one dimension and their size in the second dimension (in a direction 90 degrees from the first one). Because it is unlikely that two protein molecules will have the same charges and sizes, 2-D electrophoresis separates molecules more effectively than 1-D electrophoresis. GENOMICS, BIOINFORMATICS, BIOLOGICAL DATABASES AND PROCESSING OF BIOLOGICAL DATA. High-throughput technologies, such as DNA sequencing, microarrays, proteomics and structural genomics are sources of high amount of information related to molecular biology. DNA sequencing in combination with DNA cloning into vectors enables determination of the sequences of whole genomes. The study of organism’s entire genomes is called genomics. The first genome of a cellular organism to be sequenced was that of Haemophilus influenzae (1.8 Mb) in 1995, and since then genomes are being sequenced at a rapid rate. Among the first sequenced genomes, there were mainly those of model organisms, such as yeast Saccharomyces cerevisiae (20 Mbp), the fruit fly Drosophila melanogaster (130 Mbp), the roundworm Caenorhabditis elegans (98 Mbp), and the zebrafish Danio rerio or mouse-ear cress Arabidopsis thaliana (157 Mbp). Up to now, the genomes of more than 80 eukaryotic organisms including the mouse Mus musculus, the cat Felis catus, the dog Canis lupus familiaris, the horse Equus caballus or the cattle Bos taurus and of hundreds of prokaryotes have been sequenced. A rough draft of the human genome (aprox. 3.2 Gbp) was completed by the Human Genome Project in 2001. Over the past few decades rapid development in molecular research technologies has combined with the development in information technologies to produce, store and process large data sets. Application of information technology to the field of molecular biology is called bioinformatics. It uses mathematical and computing approaches to increase the understanding of biological processes. Bioinformatics entails the creation and advancement of databases, algorithms, computational and statistical techniques and theory to solve formal and practical problems arising from the management and analysis of biological data. Bioinformatic approaches can be used to analyze and align DNA and protein sequences, or to create and view 3-D models of protein structures. Various institutions all around the world deal with the creation and advancement of databases of nucleic acid and protein sequences. Institutions that carry out integrated databases of NA and protein sequences: The European Bioinformatics Institute (EBI) is a non-profit academic organisation, the part of the European Molecular Biology Laboratory (EMBL). This Institute manages databases of biological data including NA, protein sequences and macromolecular structures available at http://www.ebi.ac.uk. The National Centre for Biotechnology Information (NCBI) offers a free access to the genetic sequence database GenBank at http://www.ncbi.nlm.nih.gov/Genbank/. The Universal Protein Resource (UniProt) is a project operated by the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). UniProt web (www.uniprot.org) is source of protein sequences, domains structures, post-translational modifications. As science moves towards understanding biology at the systems level, access to large data sets of many different types have become crucial. There is growing need to collect and store all this information in ways that allow its efficient retrieval and exploitation. The fastgrowing amount of science information require tools to easily access and process these data, including publication search, software tools for sequence alignment, gene finding, protein structure prediction, prediction of gene expression and protein interactions, etc. In addition to the afore-mentioned databases, many organisations offer resources and expertise to facilitate the work with scientific information. NCBI was established in 1988 63 and it creates public databases, conducts research in computational biology, develops software tools for analyzing genome data, and disseminates biomedical information. Entrez search page of NCBI enables search in various databases. NCBI databases, including catalogue of human genes and genetic disorders OMIM (Online Mendelian Inheritance in Man) or digital archive of full-text, life sciences journal literature PubMed Central are available at http://www.ncbi.nlm.nih.gov/. EMBL-EBI focuses on developing and applying computationally intensive techniques (e.g. data mining or machine learning algorithms for gene finding, genome assembly, protein structure prediction, etc). Various software tools for nucleotide sequences or protein analyses (e.g. gene identification including their structure – exones, intrones, promotors, etc.), their translations into aminoacid sequences, etc. are available at http://www.ensembl.org or http://www.expasy.org. Comparison of sequences (or whole genomes) of different organisms, is basic tool of comparative genomics, which is the study of the relationship of genome structure and function across different biological species. Molecular biology and bioinformatics approaches are also often used in the field of phylogenetics (the study of evolutionary relatedness among various groups of organisms, e.g. species or populations). ___________________________________________________________________________ TASKS TASK 1: excursion to molecular biology laboratories Notice the instrumentation and spatial arrangement of the individual laboratories. TASK 2: demonstration of molecular biology methods Get acquainted with the most commonly used devices in the molecular biology lab, such as the thermocycler, apparatus for gel electrophoresis, or blotter. Understand the principles of the demonstrated methods (PCR, gel electrophoresis, RFLP, DNA sequencing, etc.). View and try to interpret the outputs of some of the methods (electrophoresis and RFLP). 9.3. Care of laboratory animals and animal experiments The terms animal testing or animal experimentation means the use of non-human animals in scientific experimentation. Animals have been used throughout the history of scientific research. Louis Pasteur demonstrated the germ theory of disease by infecting sheep with anthrax. Ivan Petrovich Pavlov used dogs to describe classical conditioning. Insulin was first isolated from dogs in 1922, and revolutionized the treatment of diabetes. The russian dog, Laika, became the first of many animals to orbit the earth etc. Since the 18th century, medicine developed thanks to experiments on domestic or wild animals, but also anti-vivisection organisation was established in 1875. Vivisection (from Latin "vivo" = live and "section" = cutting), refers to inhuman and cruel experiments on animals. In 1876, the first law protecting experimental animals was established in England. Since the 20th century, laboratory animals have been designated for experiments were produced in many countries all over the world. The research is conducted by universities, medical schools, pharmaceutical companies, defence establishments, and commercial facilities that provide animal-testing services to industry. It includes pure research i.e. in genetics, developmental biology, behavioural studies, as well as applied research such as biomedical research, xenotransplantations, drug testing and toxicology tests, including cosmetics testing. Animals are also used for education, breeding, and defence research. Scientists and government state that animal testing should cause as little suffering to animals as possible, and that animal testing can be performed only 64 if it is argued that it is scientifically justified, and that alternatives, including non-animal experiments, have been considered, and that the experiments are not unnecessarily duplicative. The "three Rs" are guiding principles for the use of animals in research in most countries: Reduction refers to methods that enable researchers to obtain comparable levels of information from fewer animals, or to obtain more information from the same number of animals. Replacement refers to the preferred use of non-animal methods (tissue or cell cultures, immunologic methods, mathematic models, video and films) over animal methods whenever it is possible to achieve the same scientific aim. Refinement refers to methods that reduce or minimize potential pain, suffering or distress, and enhance animal welfare for the animals still used. LABORATORY ANIMALS Invertebrates. Although the many more invertebrates than the vertebrates are used, these experiments are largely unregulated by law. The most used invertebrate species are Drosophila melanogaster (fruit fly) and Caenorhabditis elegans (nematode worm). These animals offer great advantages over vertebrates, including their short life cycle and multiple offspring. However, the lack of an adaptive immune system and their simple organs prevent worms from being used in medical research such as vaccine development. Similarly, flies are not widely used in applied medical research, as their immune system differs greatly from that of humans, and diseases in insects can be very different from diseases in more complex animals. Vertebrates. Mice are the most commonly used vertebrate species because of their size, low cost, ease of handling and fast reproduction rate. Mice are widely considered to be the best model of inherited human diseases. With the advent of genetic engineering technologies, genetically modified mice can provide models for a range of human diseases. Rats are also widely used for physiology, toxicology and cancer research, but genetic manipulation is much harder in rats than in mice, which limits the use of these rodents in basic science. Other rodents commonly used are guinea pigs, hamsters, and gerbils. Other laboratory animals are rabbits and African clawed frogs. The main fish species the zebrafish (Danio rerio) is used in ecotoxicological studies. Cats are most commonly used in neurological research. Dogs are widely used in biomedical research, testing of toxicity of drugs, and education. Beagles particularly are commonly used as models for human diseases in cardiology, endocrinology, and bone and joint studies. Non-human primates are used in toxicology tests, studies of AIDS and hepatitis, studies of neurology, behaviour and cognition, reproduction, genetics, and xenotransplantation. Most of the nonhuman primates used are Rhesus monkey (macaques), marmosets, spider monkeys, squirrel monkeys, baboons and chimpanzees. Laboratory animals (vertebrates) can be divided according to bacterial colonisation: Germ-free animals are animals that have no microorganisms living in or on them. Germ-free animals are born in aseptic conditions, removed from the mother by Caesarean section. Such animals are raised within germ-free isolators in order to control their exposure to viral, bacterial or parasitic agents. Germ-free animals are used in the study of probiotic research and other animal research requiring careful control of outside contaminants that can affect the experiment. Gnotobiotic animal (from Greek roots gnotos = known and bios = life) is an animal in which only certain known strains of bacteria and other microorganisms are present. They are 65 reared in the laboratory, exposed only to those microorganisms that the researchers wish to be present in the animal. Specified Pathogen Free (SPF) animals are guaranteed free of particular pathogens. Use of SPF animals ensures that specified diseases do not interfere with an experiment (e.g. absence of respiratory pathogens such as influenza is desirable when investigating a drug's effect on lung function). Conventional animals are animals with unknown and uncontrolled bacterial colonisation or animals controlled for agents transmissible to humans and domestic animals. In accordance with Act No 246/1992 Coll., on the protection of animals against cruelty, as amended (Animal Welfare Act), the protection of animals and animal welfare in the Czech Republic (CR) is the responsibility of the Ministry of Agriculture (MoA) which provides the organisation background necessary for activities performed by the Central Commission for Animal Welfare (CCAW). Legal regulations of management and inspections of animal experiments include also the Regulation No 207/2004, on the protection, breeding, and use of experimental animals and the relevant legal regulations of the European Community. The supervision over these matters is conducted by Regional Veterinary Administrations’ inspectors in 13 regions and the Municipal Veterinary Administration in Prague. When conducting animal experiments, the significance of the research and the reasons why animal experiments are required must be explained. Animal experiments must be conducted based on scientific reasons. Animal experiment protocol must be drafted beforehand and must be approved by a competent institution. Reasons for experimenting on animals: to verify a scientific hypothesis to establish a diagnosis, to prevent and cure diseases to study animal reactions to discover environmental damage to prove the safety of substances and products or to examine their effects against pests to manufacture sera, vaccines and medicines to preserve or reproduce live material for scientific purposes for the educational use in high schools, universities, postgraduate studies etc. There are three types of facilities manipulating with laboratory animals: breed facilities that breed laboratory animals, supplier facilities that supply laboratory animals and user facilities that can use animals for experiments. All these facilities must be certificated for their activity by Central Commission for Animal Welfare. User facilities designed for performing experimental procedures on animals or for analyzing physiological functions should have appropriate equipment and should be constructed to prevent animals escaping and to enable easy cleaning and disinfection to prevent contamination by excrements and blood. Clean, hygienic conditions should be maintained at all times, and every effort made to organise the laboratory to assure that even if a laboratory animal escapes, it can be easily recaptured. Care and management of laboratory animals: At facilities, laboratory animals must be correctly cared for consideration of animal welfare while also assuring the safety of the researcher(s) and animal technicians. In this respect, the individual characteristics such as species, strain, sex, or age of animals should be taken into consideration. The inherent physiology, ecology and behaviour of the animals should be maintained, minimizing stress as much as possible. The animal must be supplied with suitable nutritional, uncontaminated food and water every day. Cage environment and animal room 66 environment must have optimal temperature, humidity, lighting etc. according to the animal species. Laboratory animal health management must be conducted scientifically, including microbiologic control of the rearing environment or preventing laboratory animals from suffering injuries. Reports of all animal experiment procedures and results have to be drawn up, containing the number of laboratory animals used, information whether any changes were made to the protocol, etc. Qualification. Only a professionally qualified person with university education in the medical, veterinary, or other biological field can carry on management and control of animal experiments. This professionally qualified person must meet the qualification requirements appointed by the relevant legal regulations. Qualifications required are defined by Act No 246/1992, on the protection of animals against cruelty, as amended, and by Regulation No 207/2004, on the protection, breeding, and use of experimental animals. Managers of animal experiments must obtain a Certificate of competency according to § 17 of the Act No. 246/1992 coll. from the Central Commission for Animal Welfare. Acquisition of the certificate requires professional theoretical knowledge of the conditions for breeding, and use of experimental animals; of keeping, recording, archiving, and protection of data connected with the breeding and use of experimental animals in accordance with European Community regulations; of the ethical principles of work with animals; of the biology and genetics of laboratory animals and of the basics of their anatomy and physiology; of the nourishment and zoohygiene, and, among others, of gnotobiology and illnesses of experimental animals and of diagnostic methods, etc. Among the professional practical skills required, there are methods of breeding and work on experimental animals, their protection, the search for and application of alternative methods, experimental operations on laboratory animals, treatment of animals, use of animals in experiments, experiments on livestock, experiments on wild animals, biological experimental models, alternative methods, safety and health in connection with the work with animals. ___________________________________________________________________________ TASKS TASK 1: Excursion into the user facilities Notice rooms separated for keeping animals, feed storage, storage of bedding materials, storage of equipment necessary for usage of animals for experiments. Draw scheme of user facilities. 67 10. Transport of substances, osmosis The cell is an open system with a flow of energy, substances and information. The transport of substances is regulated by selectively permeable cell membranes (e.g. plasma membrane, nuclear envelope, tonoplast or membranes surrounding various organelles). They are usually formed by a phospholipid bilayer. The cell membrane contains many integral membrane proteins (e.g. ion channels) which are involved in different cellular processes such as transport, cell adhesion, and cell signalling. SIMPLE (FREE) DIFFUSION is a passive transport of molecules (e.g. carbon dioxide, oxygen, ethanol, urea, water, oxygen, ethanol, water) in one direction from a region of higher concentration to one of lower concentration by random molecular motion until equilibrium is reached. The speed of diffusion can be accelerated by a temperature increase. OSMOSIS is the diffusion of a solvent (frequently water) through a semi-permeable membrane (impermeable to organic solutes with large molecules, such as polysaccharides, while permeable to water and small, uncharged solutes), from a solution of low solute concentration (high water potential) to a solution with high solute concentration (low water potential), up a solute concentration gradient. Solvent diffuses from the less-concentrated (hypotonic) to the more-concentrated (hypertonic) solution, and it tends to reduce the difference in concentrations. The turgor pressure of a cell is largely maintained by osmosis. If a cell is placed into a solution which is more concentrated than its cytoplasm, it will shrivel, and if it is placed in a less concentrated solution, it will expand and/or burst. The animal cell: Hypotonic solution – water enters the cell, the cell increases its volume, the cell membrane stretches until it breaks = plasmoptysis or osmotic lysis (for erythrocytes, term osmotic haemolysis is used). Hypertonic solution – water goes out of the cell, the cell shrinks to be a star shaped = plasmorhisis The plant cell: Hypotonic solution – water runs inside only to a certain extent, because the cell wall restricts the expansion ( = turgor increase) Hypertonic solution – water runs outside, cytoplasm and vacuoles decrease their volume and cell membrane separates from the cell wall = plasmolysis FACILITATED DIFFUSION (or FACILITATED TRANSPORT) is a passage of polar molecules (aminoacids, nucleotides, sugars) or charged ions (H+, Na+, K+, Ca2+, Mg2+, Cl-) across a biological membrane through specific transmembrane transport proteins. All polar molecules and charged ions are dissolved in water and can’t diffuse freely across cell membranes due to the hydrophobic nature of the lipids that make up the lipid bilayers. They should be transported across membranes by transmembrane channels that can open and close, thus regulating the flow of ions or small polar molecules. Larger molecules are transported by transmembrane carrier proteins that change their conformation as the molecules are carried through. ENDOCYTOSIS is a process where cells absorb material (molecules such as proteins) from the outside by engulfing it with their cell membrane. It is used by most of the cells of the body because many substances important to them are large polar molecules, and thus cannot pass through the hydrophobic cell membrane. Pinocytosis (literally, “cell-drinking”) describes the uptake of solutes and single molecules such as proteins. 68 Phagocytosis (literally, “cell-eating”) is the process by which cells ingest large objects, such as cells which have undergone apoptosis, bacteria, or viruses. The cell membrane folds around the object, and the object is closed into a large vacuole (a phagosome). Receptor-mediated endocytosis (or clathrin-dependent endocytosis), is a process by which cells internalize molecules by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to the molecules being internalized. EXOCYTOSIS is the process by which a cell directs secretory vesicles out of the cell membrane. These membrane-bound vesicles contain soluble proteins to be secreted to the extracellular environment, as well as membrane proteins and lipids that are sent to become components of the cell membrane. __________________________________________________________________________________________ TASKS TASK l: Simple plasmolysis (plant cell in hypertonic solution) NP: onion, 1 % neutral red; 1M KNO3 Put the inner epidermis of an onion onto the slide and stain it with 1 % neutral red. Add 1M KNO3 and observe the plasmolysis. Draw your observation and write a conclusion. TASK 2: Deplasmolysis Add distilled water to the specimen from the previous task and observe the reverse process (cytoplasm and vacuoles increase their volume). Some cells are irreversibly damaged. TASK 3: Spasm plasmolysis (plant cell in hypertonic solution) NP: onion, 1 % neutral red; 1 % CaCl2, 1M KNO3 Stain the epidermis of an onion with 1 % of neutral red; add 1 % CaCl2 and 1M KNO3. Observe the unequal separation of the cytoplasmic membrane from the cell wall caused by the increased cohesion of cytoplasmic membrane. Fig. 48: Osmosis in epidermis of onion: A – simple plasmolysis, B – spasm plasmolysis. Cytoplasm Tonoplast Vacuole A B TASK 4: Turgor (plant cell in hypotonic solution) NP: pollen grains, H2O Put pollen grains on the slide and observe them using microscope. Then add water, cover with a cover glass and observe again. Write your observation. A Cytoplasm outpouring B Fig. 49: Osmosis: A – pollen grain in isotonic solution, B – pollen grain in hypotonic solution. 69 TASK 5: Macroscopic observation of osmotic haemolysis (blood in hypotonic solution) NP: blood, H2O, physiological solution Take two tubes and add 1 ml of blood into each of them. Then add 3 ml of physiological solution into one of them and 3 ml of water into the second one. Gently mix and compare both tubes. Perform the “reading test”. Write down the result of your observation. TASK 6: Microscopic observation of osmotic haemolysis NP: blood, H2O Put a small drop of blood on the slide and cover with a cover glass. Add water to one edge of the cover glass and let it suck in the part of the specimen. Then immediately suck the water off using a filter paper. Water causes haemolysis (rupture of erythrocytes) observed on interface of water and blood. TASK 7: Plasmorhisis (animal cell in hypertonic solution) NP: blood, 1M KNO3 Put a drop of blood on the slide, add 1M KNO3 and cover with a cover glass. You can observe shrunk erythrocytes (star-shaped) because of water escaping from them. Draw them and write a conclusion. Ruptured erythrocyte Erythrocyte Shrunk erythrocyte Erythrocyte B A Fig. 50: Osmosis. A – haemolysis of erythrocytes, B – plasmorhisis of erythrocytes. TASK 8: Phagocytosis PP: phagocyting leucocytes stained by Pappenheim method Find and draw leucocytes with phagocyted particles. You can count phagocytic activity (PA): PA = number of phagocytic cells / total number of cells. Nucleus Phagocyted particles Leucocyte Fig. 51: Leucocytes with phagocyted particles. 70 11. Cell growth and reproduction CELL CYCLE is the series of events that take place in a eukaryotic cell leading to its replication. These events can be divided in two periods: the interphase during which the cell prepares itself for cell division and the mitotic (M) phase during which the cell splits itself into two identical cells (daughter cells). INTERPHASE is divided into three phases: G1 (first gap), S (synthesis) and G2 (second gap). In G1 phase, the cell grows by producing proteins and cytoplasmic organelles. In the S phase, chromosomes are duplicated (replicated chromosomes start to have two sister chromatids). In the G2 phase, the cell continues the production of proteins and cell organelles. Differentiated cells (e.g. nerve and muscle cells) stop their division and enter the latent phase G0. MITOSIS (M phase) is the process in which somatic eukaryotic cell separates the chromosomes in its cell nucleus, into two identical sets to form two daughter nuclei. This phase includes mitosis (also karyokinesis) – division of chromosomes in two daughter nuclei and cytokinesis division of cytoplasm, organelles and cell membrane into two daughter cells. However, there are many cells where mitosis and cytokinesis occur separately, forming single cells with multiple nuclei. The process of mitosis is complex and highly regulated. Mitosis is important for maintaining the chromosomal set; each cell formed receives chromosomes that are equal in number and composition to the chromosomes of the parent cell. Mitosis is divided into five phases: prophase, prometaphase, metaphase, anaphase and telophase. Prophases – the genetic material (DNA) in the nucleus is associated with proteins to form chromatin. In prophase, the chromatin condenses into compact mitotic chromosomes, visible through a light microscope. The chromosome is composed of two sister chromatids, bound together at the centromere by the cohesion complex. Close to the nucleus, there are two centrosomes (duplicated before mitosis) made of a pair of centrioles (centrioles are missing in plant cells). The centrosome is the coordinating centre to form the mitotic spindle by the polymerizing protein tubulin. The molecular motor proteins then push the centrosomes along these microtubules to opposite poles of the cell. Although centrosomes help organise microtubule assembly, they are not essential for the formation of the spindle, since they are absent in plants. During this phase, nucleolus disappears and each chromosome forms two kinetochores (complexes of proteins) at Chromatid Kinetochore microtubule Centrosome Centriole s Astral microtubule Polar microtubule Equatorial plane the centromere, one attached at each chromatid. 71 Fig. 52: Mitotic spindle inside the animal cell during metaphase. Prometaphases – the nuclear envelope disassembles and kinetochore microtubules attach to the kinetochore of chromatids. Although the kinetochore structure and function are not fully understood, it is known that it contains molecular motor (dynein). When a microtubule connects to the kinetochore, the motor activates, using energy from ATP first to position the chromosomes into the equatorial plane (in metaphase) and subsequentially to pull the chromatides toward the centrosome (in anaphase). This motor activity, coupled with polymerisation and depolymerisation of microtubules, provides the pulling force necessary to separate the chromosome's two chromatids. Metaphase – chromosomes are lined up in the equatorial plane (or metaphase plate) by pulling powers generated by kinetochores. Unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. The signal creates the mitotic spindle checkpoint. Anaphase can be divided into two stages. Early anaphase is usually defined as the separation of the sister chromatids. The proteins (cohesins) that hold sister chromatids together are cleaved by enzyme (separase) allowing them to separate. These sister chromatids, which have now become distinct sister chromosomes, are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. Late anaphase involves the elongation of the non-kinetochore microtubules pushing the centrosomes apart to opposite ends of the cell. Telophase is a reversal of prophase and prometaphase events. The non-kinetochore microtubules continue to lengthen, elongating the cell even more. Corresponding sister chromosomes attach at opposite ends of the cell. A new nuclear envelope forms around each set of separated sister chromosomes, using fragments of the parent cell's nuclear membrane. Both sets of chromosomes, now located in new nuclei, unfold (decondense) and become unstainable and thus invisible. The nucleolus is formed. Cytokinesis is a separate process that begins at the same time as telophase (sometimes it starts in anaphase). Cytokinesis is technically not even a phase of mitosis, but rather a separate process, necessary for completing cell division. In animal cells, a cleavage furrow containing a contractile ring (made of actin and myosin II) develops where the metaphase plate used to be. In both animal and plant cells, cell division is also driven by vesicles derived from the Golgi apparatus, which move along microtubules to the middle of the cell. In plant cells, vesicles move to the centre of the phragmoplast and develop into a cell wall, separating the two cells. The phragmoplast is a microtubule structure typical for higher plants, whereas some green algae use a phycoplast microtubule array during cytokinesis. Nucleus Nucleus Phragmoplast Contractile ring Microtubule Cell plate Vesicles Microtubule 72 Plant cell Animal cell Fig. 53: Cytokinesis in plant and animal cells. MITOTIC INDEX determines frequency of mitosis in plant or animal tissues (or in cell cultures). MI (%) = M/N x 100 where M = number of mitotic cells, N = total number of cells. 73 MITOTIC DISORDERS can be spontaneous (with unknown cause) or can be caused by different mutagens (substances with genotoxic activity e.g. hydroquinone) and can lead to: Fragmentation of chromosomes in prophase Anaphase bridge formation Defective anaphase with damaged chromosomes Although errors in mitosis are rare, the process may go wrong, especially during early cellular divisions in the zygote. Such mitotic errors can be especially dangerous to the organism because future offspring of this parent cell will carry the same disorder (for more information see Chapter 15.1.). 11.1. Mitosis in plant cell Mitosis in plant cells is much better observable compared to animal cells. Cells are larger and individual chromosomes and even microtubules of mitotic spindle can be perfectly observed under light microscope. ___________________________________________________________________________ TASKS TASK 1: Mitosis in onion rootlet cells NP: onion rootlet stained with acetorcein Compress an onion rootlet under a cover glass with your finger (compression preparation) and observe the particular mitotic phases. Staining procedure: The base of onion is sunk in water to let roots grow. When roots start to grow, their ends (about 2-3 mm) are cut and fixed in acetic acid and 96 % ethanol (in ratio 1:3) for 20 min. Then roots are macerated in hydrochloric acid and ethanol (in ratio 1:1) for 10 min., washed in distilled water for 10 min. and finally stained with acetorcein for 10 min. Telophase Monaster Early anaphase Prophase Metaphase Late anaphase Interphase Fig. 54: Mitosis in onion rootlet cells. 74 TASK 2: Mitotic phases in permanent preparation PP: onion (and/or bean) rootlet - “kořínek cibule, kořínek bobu” Observe 30 mitotic figures (cells in mitosis) and determine mitotic phases. Which of them is the most frequent and why? TASK 3: Defects of mitosis NP: onion rootlets treated with hydroquinone and stained with acetorcein. Prepare compression preparation of the onion rootlet and observe defects of mitosis. C B A Fig. 55: Defects of mitosis: A – chromosome fragmentation in prophase, B – anaphase bridge, C –defective anaphase in cell with damaged chromosomes. 11.2. Mitosis in animal cell Mitosis in animal cells is not so easily observable compared to plant cells. Mitotic cells are larger and brighter compared to interphase cells, with rounded edges and with mitotic figure instead of nucleus. ___________________________________________________________________________ TASKS TASK 1: Mitosis in histologic section of small intestine PP: small intestine of laboratory rat - “mitóza střevo”, stained with haematoxylin-eosin. Observe the cross-section of the small intestine under small and then use a higher magnification. In the intestinal villi you can find mitotic dividing cells. Small intestine Intestinal villus Anaphase Metaphase B Fig. 56: Mitosis in intestinal villi of small intestine. 75 TASK 2: Mitosis in histologic section of uterus PP: uterus of laboratory rat - “mitóza uterus”, stained with haematoxylin-eosin Observe the cross-section of the uterus under small and then higher magnification. Mitotic dividing cells can be found in uterine mucosa. Mucosa of uterus Uterus Anaphase Telophase Fig. 57: Mitosis in mucosa of uterus. TASK 3: Mitosis in histological section of testes PP: testes of laboratory rat - “varle krysa”, stained with haematoxylin-eosin Observe the specimen under small and then larger magnification. Mitotic dividing cells are inside the seminiferous tubules on the periphery. Note: do not mistake mitotic cells for those dividing by meiosis. Testes Seminiferous tubules of testes Anaphase Fig. 58: Mitosis in seminiferous tubules of testes. TASK 4: Mitotic index PP: specimen from cell culture - “TK mitóza” Observe the cells in five visual fields; count how many cells undergo mitosis and how many of them are in interphase and calculate mitotic index. In this specimen, you can also find monaster (cell in metaphase) and the anaphase bridge. 76 12. Movement and irritation 12.1. Movement BROWNIAN MOLECULAR MOTION (named after the Scottish botanist Robert Brown) is the random zigzag movement of particles suspended in a liquid or gas. This motion is caused by collisions of the particle with much smaller liquid molecules (which are in random thermal motion). CELLULAR MOVEMENT is active process that includes intracellular transport of substances, the changing of cell shape or locomotion. Each movement is mediated by cytoskeletal fibers - microfilaments (actin filaments) or microtubules. Energy is released from ATP by molecular motors (dynein or kinesin associated with microtubules, myosin I or myosin II associated with microfilaments). AMOEBOID MOVEMENT is a crawling-like type of movement in which the cell forms temporary cytoplasmic projections called pseudopodia (“false feet”). Microfilaments and myosin (type I) are involved in this process. This type of movement is observed in amoebae (e.g. Amoeba proteus). Apart from amoeba, other examples are cellular slime moulds (e.g. Dictyostelium discoideum), and human cells, particularly macrophages (including Kupffer cells of liver), monocytes or neutrophils. CILIARY AND FLAGELLAR MOVEMENT. Although cilia and flagella are structurally the same, they differ in number, size and their beating pattern. In the case of flagella (e.g. the tail of a sperm) the motion is propeller-like. In contrast, the beating of cilia consists of coordinated back-and-forth cycling of many cilia on the cell surface. Thus, motile flagella serve for the propulsion of single cells (e.g. swimming of protozoa and spermatozoa), and cilia for the transport of fluids (e.g. transport of mucus by stationary ciliated cells in the trachea). However, cilia are also used for locomotion (through liquids) in organisms such as Paramecium. Typically, cells possess one or two long flagella, whereas ciliated cells have many short cilia (for example, the mammalian spermatozoon has a single flagellum, while huge numbers of cilia cover the surfaces of cells in mammalian respiratory passages) . Both cilia and flagella are encased within the cell's plasma membrane. All eukaryotic cilia and flagella are similar in their organisation, possessing a central bundle of microtubules (made of tubulin); called the axoneme, in which nine outer doublet microtubules surround a central pair of microtubules (9 + 2 arrangement). Each doublet microtubule consists of A tubule (contains 13 protofilaments) and B tubule (contains 10 protofilaments). At its point of attachment to the cell, the axoneme connects with the basal body, cylindrical structure which contains nine triplet microtubules. Radial spoke Central microtubule Dynein Microtubule doublet (A and B unit) A B 77 Fig. 59: Cross-section of cilia/flagella. The basal body plays an important role in initiating the growth of the axoneme. The axoneme is held together by protein cross-links (bridges between central pair of microtubules and A tubule of the outer doublets). Cilia and flagella possess an active ATPase that is associated with the dynein arms. Binding and hydrolysis of ATP causes walking of dynein arms extending from one doublet toward the neighbouring doublet and generates a sliding force in the axoneme. Bacterial flagella are made up of the protein flagellin and have completely different structure, compared to eukaryotic flagella. MUSCLE CONTRACTION occurs when a muscle fiber (myofibril) iside of the muscle cell generates tension through the action of actin and myosin proteins. Muscle cells are surrounded by sarcolemma, that consists of a plasma membrane, and of the outer coat made up of a thin layer of polysaccharide material that contains numerous thin collagen fibrils. Basic contractile units of cardiac and skeletal muscles myofibrils are called sarcomeres. While under tension, the muscle may lengthen, shorten or remain the same. Locomotion in higher animals is possible only through the repeated contraction of many muscles at the correct times. The contraction is controlled by the central nervous system (CNS), which is composed of the brain and spinal cord. Voluntary muscle contractions are initiated in the brain, while the spinal cord initiates involuntary reflexes. There are three general types of muscle tissues: skeletal muscle responsible for movement, cardiac muscle responsible for pumping blood and smooth muscle responsible for involuntary contractions in the blood vessels, gastrointestinal tract, uterus and other areas in the body. Skeletal and cardiac muscles are called striated muscles because of their striped appearance under a microscope which is due to actin and myosin composition. Striated muscle contraction follows these steps: 1) Action potencial (nerve signal) originated in CNS transmits action potential down its own axon (projection of nerve cell), activates voltage-gated calcium channels on the axon, and calcium rushes in. 3) Calcium causes that vesicles transported along microtubules in nerve cells release neurotransmitter acetylcholine into synaptic cleft between neuron and skeletal muscle fiber. 4) Acetylcholine diffuses across synapse, activates receptors of muscle resulting in opening of sodium/potassium channel - sodium rush in and potassium rush out. 5) Action potential spreads through muscle fiber’s network of T tubules (invaginations of membrane) and depolarizes the inner part of the muscle and thus activates voltage-gated calcium channels of sarcoplasmic reticulum (SR, special endoplasmic reticulum) to release calcium. 7) Calcium binds to protein troponin C that blocks actin filament to bind with myosin. Troponin C changes its conformation and thus modulates other protein (tropomyosin) allowing it to move and unblock the binding sites on actin for myosin. 8) Myosin (a fiber made of about 300 molecular motors myosins II) binds to uncovered binding sites on actin (microfilament), hydrolyzes ATP to obtain energy and move along actin resulting in shortening of sarcomere and muscle contraction. Z disc Myosin Actin 78 Z disc Fig. 60: Sarcomere – the basic contractile unit in muscle (from one to the other Z disc). Muscle relaxation: muscle contraction is stopped when there is no action potential, calcium is pumped to SR by the calcium pump, tropomyosin changes its conformation back to its previous state to block the binding sites of actin. Smooth muscle contraction is similar to striated skeletal muscle contraction, but different proteins are involved. The contraction is slower, less specialized and directed by signals e.g. adrenalin, serotonine and prostaglandine. In vertebrates, calcium is released from SR and binds to protein calmodulin that activates myosin-light chain kinase to form calcium-calmodulin-myosin light chain kinase complex that phosphorylates myosin to initiate contraction and activate myosin ATPase. In invertebrates, calcium directly activates phosphorylation of myosin and then generates force. ___________________________________________________________________________ TASKS TASK 1: Brownian motion Place a drop of ferric oxide suspension onto a slide and cover it. Observe one small moving particle and draw trajectory of its movement. What is the principle of this movement? TASK 2: Amoeboid movement NP: hay infusion Observe movement of amoeba from the hay infusion. First, they are irritated and round, after some time they start to show protrusions (pseudopodia) and move. Pseudopodia are homogenous, cytoplasm is granulated. What is the principle of this movement? TASK 3: Flagellar movement NP: sperm (or hay infusion) Observe the flagellar movement of sperm (movement forward and rotation around axis) or movement of flagellar protozoans from hay infusion. If you want to slow them down, suck off water from the space between the two glasses using a filter paper. A B C Fig. 61: Types of movements: A – Brownian movement, B – amoeboid movement, C – flagellar movement. TASK 4: Ciliary movement NP: hay infusion, detritus from aquarium Observe ciliary movement of unicellular ciliates from hay infusion or ciliary movement on the surface of some multicellular organisms (flatworm) from aquarium. What is the principle of a ciliary movement? TASK 5: Cells with cilia 79 PP: cells with cilia - “řasinkový epitel, žába”, stained with haematoxylin-eosin Observe and draw cells of cylindrical or conical shape with visible nucleus and with cilia localized at the wider base of the cell. TASK 6: Structure of striated muscle PP: striated muscle from an insect leg - “příčně pruhovaný sval”, stained with Heidenhain haematoxylin Observe the striated muscle. Striation is caused by different staining of muscle fibers. What is the principle of a muscle contraction? Myosin Cilium Actin A B Fig. 62: A – cell with cilia, B – striated muscle of insect. 12.2. Irritation TAXIS are directional movements by an organism towards or away from the stimulus. If the organism moves towards the stimulus, then the taxis are positive. If the organism moves away from the stimulus, then the taxis are negative. Many types of taxes have been identified and named using prefixes to specify the stimulus that induces the response: phototaxis (stimulation by light) chemotaxis (chemicals) oxygenotaxis (oxygen) thermotaxis (temperature changes) hydrotaxis (moisture) thigmotaxis (physical contact) barotaxis (pressure) galvanotaxis (electrical current) geotaxis (gravity) anemotaxis (stimulation by wind) rheotaxis (fluid flow) TROPISM (from Greek, tropos = to turn) is the directional growth or turning movement of a biological organism (usually a plant) or its part in response to an environmental stimulus. Tropisms are usually named according to the stimulus involved and may be either positive (towards the stimulus) or negative (away from the stimulus). These include: phototropism (response to lights or colors of light) gravitropism or geotropism (gravity) thigmotropism (touch or contact) hydrotropism (moisture or water) heliotropism (sunlight) thermotropism (temperature) 80 NASTIC MOVEMENTS are plant movements that are caused by an external stimulus, such as light or temperature, but are directionally independent of its source, unlike tropisms. Nastic movements occur as a result of changes in water pressure within specialized cells or differing rates of growth in parts of the plant. thermonasty (opening and closing of crocus flowers following an increase or decrease in temperature) photonasty (opening and closing of evening primrose (Oenothera) flowers on exposure to light or dark ) haptonasty (leaf movements of the Venus flytrap (Dionaea muscipula) following a tactile stimulus, rapid collapse of the leaflets of the sensitive plant Mimosa pudica) nyctinasty (“sleep movements”, where the leaves or flowers of some plants adopt a different position at night) hydronasty (in response to a change in the atmospheric humidity) chemonasty (in response to a chemical stimulus) ___________________________________________________________________________ TASKS TASK 1: Chemotaxis in ciliates NP: hay infusion Put two drops of hay infusion onto the slide (do not cover). Connect both drops using skewer to form a thin bridge. Then add several crystals of salt (NaCl) to one of the drops. Observe negative chemotaxis. + NaCl Hay infusion TASK 2: Oxygenotaxis in ciliates NP: hay infusion Cover a drop of hay infusion with cover glass and form air bubbles under it. You can observe positive oxygenotaxis. The same effect can be observed at the edges of cover glass. Air bubble 81 13. Reproduction and development Reproduction is the biological process by which new individual organisms are produced. Reproduction is a fundamental feature of all known life; each individual organism exists as the result of reproduction. The known reproductive strategies are broadly grouped into two main groups: sexual and asexual. ASEXUAL REPRODUCTION Asexual reproduction is the process by which an organism creates a genetically identical copy of itself without a contribution of genetic material from another individual. Bacteria divide asexually via binary fission; hydras (invertebrates of the order Hydroidea) and yeasts are able to reproduce by budding. Plants are capable of vegetative reproduction (reproduction without seeds or spores) but can also reproduce sexually. Likewise, bacteria may exchange genetic information by conjugation. Other ways of asexual reproduction include: fragmentation and spore formation that involves only mitosis. SEXUAL REPRODUCTION Sexual reproduction is a biological process by which organisms (most animals including humans, and many plant species) create offspring that inherit one allele for each trait from each parent, thereby ensuring that offspring have a combination of the parents' genes. Offspring are produced by fusion of haploid sex cells (gametes) produced by meiosis. Fusion of gametes is referred to as fertilisation, resulting in diploid organism formation. Anisogamous species - the two sexes are referred to as male (producing sperm or microspores) and female (producing ova or megaspores). Isogamous species - the gametes are similar or identical in form, but may have different properties and then may be given other different names. For example, in the green alga, Chlamydomonas reinhardtii, there are so-called "plus" and "minus" gametes. A few types of organisms, such as some ciliates, have more than two kinds of gametes. Parthenogenesis is the growth and development of the embryo or seed without fertilization by a male. In parthenogenesis, egg cells may be produced via meiosis or mitosis oogenesis, thus being haploid or diploid, respectively. Parthenogenesis occurs naturally in some species including lower plants (where it is called apomixis), invertebrates (e.g. water fleas or aphids), and vertebrates (e.g. some reptiles, fish, and rarely sharks). Several insect species demonstrate facultative parthenogenesis and their unfertilized eggs develop into only one sex, the other sex arising from fertilized eggs. Thus, for example, in the honey bee (Apis mellifera), the males (drones) always develop from unfertilized eggs and are haploid, whereas fertilized eggs develop either into sexually mature females (queens) or into female workers (both diploid), depending on the nutrition of their respective larvae. Gynogenesis - offspring are produced by the same mechanism as in parthenogenesis, but with the requirement that the egg be stimulated by the presence of sperm in order to develop. However, the sperm cell does not contribute any genetic material to the offspring. Gynogenesis occurs e.g. in fish, flatworms or some salamander species. Androgenesis refers to development of a zygote that contains only paternal chromosomes. New individual can develop only from male gamete, or oocyte chromosomes are absent or inactivated after fertilization. Androgenesis is present only in plants. GONOCHORIST (dioecious) is an individual, that has either ovaries or testes. HERMAPHRODITE (monoecious) is an individual that has both male and female sex organs. AUTOGAMY (self-fertilization) occurs in most flowering plants, numerous protozoans, and many invertebrates. Two gametes fused in fertilization come from the same individual. 82 Organisms that reproduce through asexual reproduction tend to grow in number exponentially. However, because they rely on mutation for variations in their DNA, all members of the species have similar genomes and thus similar vulnerabilities in changing environment. Organisms that reproduce sexually yield a smaller number of offspring, however due to much higher genetic variability (and thus increased rate of evolution by natural selection), the populations of sexually reproducing organisms are able to respond readily to changing conditions. ESTROUS CYCLE comprises the recurring physiologic changes that are induced by reproductive hormones in most mammalian placental females. Humans undergo a menstrual cycle instead. Estrous cycles start after puberty in sexually mature females and are interrupted by anestrous phases. Typically estrous cycles continue until death. Phases of estrous cycle are: 1) Proestrus – one or several follicles of the ovary are starting to grow. Typically this phase can last as little as one day or as long as three weeks, depending on the species. Under the influence of estrogen the lining in the uterus (endometrium) starts to develop. Some animals may experience vaginal secretions that could be bloody. The female is not yet sexually receptive. 2) Estrus – under regulation by gonadotropic hormones, ovarian follicles are maturing and estrogen secretions have their biggest influence. Animals exhibit a sexually receptive behaviour (is "in heat," or "on heat") signalled by visible physiologic changes (lordosis reflex, in which the female spontaneously elevates her hindquarters). In some species, the vulvae are reddened. Ovulation may occur spontaneously in some species (e.g. cattle), while in others it is induced by copulation (e.g. cat). 3) Metestrus – a temporary endocrine structure called corpus luteum develops from an ovarian follicle. The uterine lining begins to secrete small amounts of progesterone. This phase may last one to five days. In some animals bleeding may be noted due to declining estrogen levels. 4) Diestrus is characterised by the activity of the corpus luteum that produces progesterone, which is needed to maintain pregnancy. In the absence of pregnancy the diestrus phase terminates with the regression of the corpus luteum to corpus albicans. 5) Anestrus refers to the phase when the sexual cycle rests. This is typically a seasonal event controlled by light exposure through the pineal gland that releases melatonin. Melatonin may repress stimulation of reproduction in long-day breeders and stimulate reproduction in short-day breeders. Anestrus is induced by time of year, pregnancy, lactation, significant illness, and possibly age. POLYESTROUS ANIMALS - go into heat several times a year (e.g. cats, cattle and pigs). It is the effect of domestication. SEASONALLY POLYESTROUS ANIMALS - have more than one estrous cycle during a specific time of the year and can be divided into: short-day breeders (sexually active in fall or winter e.g. sheep, goats, deer) and long-day breeders (sexually active in spring and summer e.g. hamsters). DIESTROUS ANIMALS - species that go into heat twice per year (most dogs). MONOESTROUS ANIMALS - have only one breeding season a year, typically in spring to allow growth of the offspring during the warm season to survive the next winter (wolves). A few mammalian species, such as rabbits, do not have an estrous cycle and are able to conceive at almost any arbitrary moment. 83 13.1. Development PHYLOGENY is the evolutionary development and history of species or higher taxa. ONTOGENY (ONTOGENESIS) is the origin and development of an individual organism from embryo to adult. INDIRECT DEVELOPMENT is a development of individuum through larva stage that differs usually from adult in food uptake strategy, environment they live in and in a body plan. In insect there are two variants: 1) Hemimetabolism (or hemimetaboly or incomplete metamorphosis) is a term used to describe the mode of development of certain insects that includes three distinct stages: the egg, nymph, and the adult stage (imago). These insect go through gradual changes; there is no pupal stage. Hemimetabolous insect groups include: Orthoptera (grasshoppers and crickets), Mantodea (praying mantises), Blattaria (cockroaches), Dermaptera (earwigs), Odonata (dragonflies and damselflies), Phasmatodea (stick bugs), Isoptera (termites), Phthiraptera (lice), Ephemeroptera (ephemeron), Plecoptera (stonefly). Hemimetabolous insects used to be further divided into two categories: paurometaboly - the nymph and the adult would live in the same environment (water, air, soil, etc.) like in case of the Orthoptera (grasshoppers and crickets) and some Hemiptera (true bugs) and heterometaboly - the nymph and adult of heterometabolous insects live in different environments, e.g. Odonata naiads live in the water and cicada nymphs underground, whereas the imagos are aerial. 2) Holometabolism (called complete metamorphosis) is a term applied to insect groups to describe the specific kind of insect development which includes four life stages: embryo, larva, pupa and imago. For example, in the life cycle of a butterfly, embryo grows within the egg, hatching into the larval stage (caterpillar), before entering the pupal stage within its chrysalis and finally emerging as an adult butterfly imago. Holometabolous insect groups include: Coleoptera (beetles), Diptera (flies), Hymenoptera (ants, bees, sawflies and wasps), Lepidoptera (butterflies and moths), Mecoptera (scorpionflies), Megaloptera (alderflies, dobsonflies and fishflies), Neuroptera (lacewings, antlions, etc.), Raphidioptera (snakeflies), Siphonaptera (fleas), Strepsiptera (twisted-winged parasites), Trichoptera (caddisflies). DIRECT DEVELOPMENT is a development of organism from fertilized egg to adult without larval stage. We can differentiate prenatal development with embryonal and foetal (fetal) stages and postnatal development. OVIPARITY - reproduction in which eggs are laid and embryos develop outside the mother's body. Most invertebrates and many vertebrates reproduce this way. VIVIPARITY - embryo develops inside the body of the mother that then gives live birth. Viviparous offspring live independently and require an external food supply from birth. The more developed form of viviparity is called placental viviparity with placental mammals as the best example. OVOVIVIPARITY - reproduction in which progeny develops from eggs retained within the mother's body but separated from it by the egg membranes. The eggs contain yolk, which provides nourishment for the developing embryo. Many insect groups, fish, and reptiles (vipers) reproduce this way. ___________________________________________________________________________ TASKS TASK 1: Budding yeast NP: suspension of yeasts Prepare a native preparation of yeast and observe the buds on the surfaces of some of the cells. 84 TASK 2: Cytological changes in vaginal mucosa during estral cycle PP: stained vaginal swab of rat in different phases of estral cycle “proestrus, estrus, metestrus, and diestrus”. Observe and draw a specimens from four different phases of estral cycle. Each phase has its typical finding: proestrus (large epithelial cells with dark nuclei), estrus (corneous cells without nuclei), metestrus (cells with large nuclei, mucus), diestrus (mucus and leukocytes). Bud Proestrus Fig. 63: Budding yeast. Estrus Metestrus Diestrus Fig. 64: Phases of estral cycle with typical findings in vaginal mucosa. TASK 3: Ontogenic development of different animal species PP: the insect with imperfect metamorphosis (chicken louse Menacanthus stramineus, Degeeriela rufa), flasks with developmental stages of holometabolous insect (gypsy moth Lymantria dispar), fish (common trout Salmo trutta), amphibian (common frog Rana temporaria), bird (domestic fowl Gallus gallus), mammal (brown rat Rattus norvegicus; human fetus Homo sapiens) A B C C D D E W D E F F Fig. 65: Ontogenic development of different animal species: A – chicken louse Menacanthus stramineus (egg, 3 nymphs, imago), B – gypsy moth Lymantria dispar (egg, larva, pupa in cocoon, imago), C – common trout Salmo trutta (egg, embryo, hatched larva with yolk sac (=alevin), adult fish), D – common frog Rana temporaria (egg, tadpole, frog with tail, frog), E – domestic fowl Gallus gallus (egg, embryo, chicken after hatching, adult), F – brown rat Rattus norvegicus (uterus in early and late stage of gravidity, fetus and adult of rat). 85 TASK 4: Cleavage of rabbit egg PP: egg surrounded by follicular cells and egg consisting of two blastomeres; photographs of egg with two polocytes and egg consisting of eight blastomeres “vajíčka králíka”. Observe permanent preparations and photographs. A C B D Fig. 66: Rabbit egg: A – surrounded by follicular cells, B – with two polocytes, C – consisting of two blastomeres, D – consisting of eight blastomeres. 13.2. Meiosis Meiosis is a process of reductional division in which the number of chromosomes per cell is cut in half. Meiosis is essential for sexual reproduction and therefore occurs in all eukaryotes (including single-celled organisms) that reproduce sexually. In animals, meiosis always results in the formation of gametes (sex cells), while in other organisms it can give rise to spores. During meiosis, the genome of a diploid germ cell undergoes DNA replication followed by two rounds of division, resulting in four haploid cells. Each of these cells contains one complete set of chromosomes (half of the genetic content of the original cell). If meiosis produces gametes, these cells must fuse during fertilization to create a new diploid cell (zygote) before any new growth can occur. Because the chromosomes of each parent undergo genetic recombination by crossing-over during meiosis, each gamete, and thus each zygote, will have a unique genetic blueprint. Together, meiosis and fertilization constitute sexuality in the eukaryotes, and generate genetically distinct individuals in populations. In all plants, and in many protists, meiosis results in the formation of haploid cells that can divide vegetatively without undergoing fertilization, referred to as spores. In these groups, gametes are produced by mitosis. Significance of meiosis: Meiosis facilitates stable sexual reproduction. Without the halving of ploidy (chromosome count), fertilization would result in zygotes that have twice the number of chromosomes as the zygotes from the previous generation. In organisms that are normally diploid, polyploidy results in developmental abnormalities or lethality. Plants, however, regularly produce fertile, viable polyploids. Recombination and independent assortment of homologous chromosomes allow for a genetic and phenotypic variation in a population of offspring and thus greater diversity of genotypes in the population. MEIOSIS I One chromosome set is referred to as “n”. In diploid cell (2n), the homologous chromosome pairs separate producing two haploid cells (n), so meiosis I is referred to as a reductional division. PROPHASE I – homologous chromosomes pairing (or synapsis) and crossing over (or recombination) occurs - the steps unique to meiosis. Leptotene (leptonema = “thin threads”) - individual chromosomes begin to condense (sister chromatids of one chromosome are connected by the synaptonemal complex). 86 Zygotene (zygonema = “paired threads”) - homologous chromosomes (each originally coming from one parent) line up with each other to form bivalents or tetrads (two chromosomes and four chromatids). Pachytene (pachynema = “thick threads”) – non-sister chromatids of homologous chromosomes may randomly exchange (crossing-over) segments of genetic information over regions of homology (sex chromosomes exchange only information over a small region of homology). Exchange takes place at sites called chiasmata and results in a recombination of information (each chromosome has the complete set of information it had before). Diplotene (diplonema = “two threads”) - synaptonemal complex degrades; homologous chromosomes separate from one another a little and uncoil a bit allowing transcription of DNA. Diakinesis (diakinesis = “moving through”) - four chromatids of the tetrads are visible, nucleoli disappear, nuclear membrane disintegrates into vesicles and the meiotic spindle begins to form. Kinetochore microtubules bind to chromatids. METAPHASE I – bivalents are randomly oriented along the metaphase plate. ANAPHASE I – kinetochore microtubules shorten and pull chromosomes toward opposing poles forming two haploid chromosomal sets, each containing only one chromosome of each type instead of original pair. Each chromosome still contains a pair of sister chromatids. Non-kinetochore microtubules lengthen pushing the centrioles apart and the cell elongates. TELOPHASE I – microtubules disappear, new nuclear membrane surrounds each haploid set, chromosomes (sister chromatids remain attached) uncoil. Cytokinesis occurs. Cells may enter a period of rest known as interkinesis (interphase II). No DNA replication occurs during this stage! MEIOSIS II Meiosis II is similar to mitosis and results in production of four haploid cells. PROPHASE II – disappearance of the nucleoli and the nuclear envelope, shortening and thickening of the chromatids, centrioles move to the polar regions and arrange spindle fibers for the second meiotic division. METAPHASE II – similar like metaphase I, but the metaphase equatorial plate is rotated by 90 degrees when compared to meiosis I. ANAPHASE II – microtubules pull the sister chromatids (now representing sister chromosomes of newly formed cells) towards opposing poles. TELOPHASE II – uncoiling and lengthening of the chromosomes and the disappearance of the spindle, nuclear envelopes reform and cleavage or cell wall formation produces a total of four daughter cells, each with a haploid set of chromosomes. Meiosis is now complete. The normal separation of chromosomes in meiosis I or sister chromatids in meiosis II is termed disjunction. When some chromosomes do not separate, it is called nondisjunction, resulting in the production of gametes which have too many or too few of a particular chromosome (aneuploidy), and is a common mechanism for trisomy or monosomy (see Chapter 15.1.). 13.2.1. Meiosis in humans 1) SPERMATOGENESIS Spermatogenesis produces mature male gametes - sperm (spermatozoa), which are able to fertilize female gamete to produce a zygote. In mammals, spermatogenesis occurs in testes and epididymis and in humans takes approximately 74 days (adult males produce an average 87 of 100-200 million sperm each day). It starts at puberty and usually continues uninterrupted until death. In the seminiferous tubules of testes, precursor diploid cell called spermatogonium divides by mitosis to produce two diploid primary spermatocytes. Each primary spermatocyte subsequently undergoes meiosis I to produce two haploid secondary spermatocytes. Secondary spermatocytes enter meiosis II to produce haploid spermatids. Cell type spermatogonium primary spermatocyte secondary spermatocyte spermatid ploidy/chromosome 2n /46 2n/46 n/23 n/23 chromatids 2 4 2 1 Process spermatocytogenesis (mitosis) spermatidogenesis (meiosis 1) spermatidogenesis (meiosis 2) spermiogenesis Spermatids start to maturate into sperm cell. The head (nucleus with condensed DNA) is capped by a vesicle called acrosome (derived from the Golgi complex) that contains enzymes important for penetrating the protective layers surrounding the egg. One of the centrioles elongates to become the tail of the sperm (flagellum). Maturation takes place under the influence of testosterone, and includes removal of unnecessary cytoplasm and organelles that are phagocyted by surrounding Sertoli cells in the testes. Sertoli cells maintain the environment necessary for sperm development and maturation, secrete substances initiating meiosis, secrete testicular fluid, secrete androgen-binding protein which concentrates testosterone, secrete hormones affecting the pituitary gland control of spermatogenesis and phagocytose residual cytoplasm left over from spermiogenesis. The resulting sperm cells are now mature but lack motility. They are transported to the epididymis in testicular fluid secreted by the Sertoli cells with the help of peristaltic contractions. In the epididymis, they acquire motility (are now referred to as spermatozoa) and become capable of fertilization. Sperm cell The process of spermatogenesis is highly sensitive to fluctuations in the environment, particularly hormones and temperature. Testosterone is produced by interstitial cells known as Leydig cells. The testes are located outside the body in a sack of skin called the scrotum. The optimal temperature is maintained at 2oC below body temperature. Dietary deficiencies (such as vitamins B, E and A), anabolic steroids, metals (cadmium and lead), x-ray exposure, dioxin, alcohol, and infectious diseases will also affect the rate of spermatogenesis. 2) OOGENESIS Oogenesis is the creation of female gamete (ovum, egg cell). The first part of oogenesis occurs in the ovarian follicle, the functional unit of the ovary. Precursor diploid cell oogonium divides by mitosis into two primary oocytes and is completed either before or shortly after birth. These cells stop at the prophase (diplotene) stage of meiosis I and lay within a protective shell of somatic cells called the follicle. In puberty, follicles begin growth and a small number enter the menstrual cycle. Primary oocytes continue meiosis I to form haploid secondary oocyte and the first polar body (polocyte) and stop at the metaphase of meiosis II until fertilization. When meiosis II is completed, haploid ootid and another polar body is created. Altogether, oogenesis results in formation of one haploid ovum and three polocytes. 88 Cell type oogonium primary oocyte secondary oocyte ootid ovum Ploidy 2n 2n n Process oocytogenesis (mitosis) ootidogenesis (meiosis I) ootidogenesis (meiosis II) Process completion third trimester (forming oocytes) stop in prophase I until ovulation stop in metaphase II until fertilization minutes after fertilization n n Oogonium 2n Primary oocyte (oocyte of I. order) 2n Mitosis 2n Spermatogonium 2n Primary spermatocyte (spermatocyte of I. order) Meiosis I Polar body n n Secondary oocyte (oocyte of II. order) Secondary spermatocyte (spermatocyte of II. order) n n Meiosis II n n n n Spermatids Polar bodies n Mature egg n n Mature sperm cells n B A Fig. 67: A – scheme of oogenesis, B – scheme of spermiogenesis. MENSTRUAL CYCLE is the cycle of physiological changes controlled by hormones occurring in reproductive-age of human females (or other primates). The average cycle length is 28 days. It may be divided into three phases: Follicular phase is stimulated by gradually increasing amounts of hormone estrogen. Follicles in the ovary begin developing and after several days one or occasionally two become dominant (nondominant follicles atrophy). The dominant follicle (Graafian follicle) releases an ovum in an event called ovulation. In luteal phase, the remains of the dominant follicle in the ovary become a corpus luteum producing hormone progesterone. Under the influence of progesterone, the endometrium (uterine lining) changes to prepare for potential implantation of an embryo to establish a pregnancy. Menstruation starts, if implantation does not occur within approximately two weeks, the corpus luteum will involute, causing drops in levels of both progesterone and estrogen. These hormone drops cause the uterus to shed its lining. ___________________________________________________________________________ TASKS TASK 1: Reproduction of chromosome number during meiosis PP: stained longitudinal sections of testes of beetle Blaps mortisaga Observe several sections of testes and try to find individual phases of meiosis (especially phases of first prophase). 89 Diakinesis Leptotene Pachytene Diplotene Zygotene Telophase Anaphase Leptotene Zygotene Pachytene Diplotene Diakinesis Fig. 68: Meiosis in longitudinal section of testes of beetle Blaps mortisaga. TASK 2: Spermiogenesis PP: rat testes “varle krysa”, stained with haematoxylin-eosin Observe specimen of rat testes and find seminiferous tubules, where spermiogenesis takes place (different stages). When you start from periphery, there are spermatogonia, small cells with nucleus rich in chromatin. More centripetally you can find primary spermatocytes (spermatocytes of I. order) and secondary spermatocytes (spermatocytes of II. order), that differ in size and in nucleus structure. Close to the centre of seminiferous tubule, there are small spermatids and mature sperm cells. Seminiferous tubules Sperm cell Spermatid Primary spermatocyte Secondary spermatocyte Spermatogony A B Fig. 69: Spermiogenesis in rat testes: A – rat testes with seminiferous tubules, B – seminiferous tubule with different spermiogenesis phases. 90 TASK 3: Different shape and size of sperm PP: sperm of rat, rabbit, horse, boar and bull - “spermie krysa, králík, kůň, kanec, býk”; picture of sperm of other animal species Observe sperm of different animal species and compare the shape of sperm head, the size of acrosome (structure on the top of head containing enzymes important for penetrating the egg) and size and number of sperm tails. Rat Crustacean perloočka Bull Rabbit Horse Boar Fish Ascaris Flatworm Fig. 70: Sperm of different animal species. TASK 4: Oogenesis PP: rat ovary - “ovarium krysa” Observe the specimen of a rat ovary and find the primary follicle (oocyte surrounded by follicular cells) and mature follicle (Graafian follicle) with multiplied follicle cells and cavities filled with fluid. Secondary follicle Primary follicle Folicular cells Oocyte Nucleus Graafian follicle Nucleolus Fig. 71: Oogenesis in rat ovary. 91 14. Influence of surroundings onto the bioplasm When all the compounds inside and outside of the cell are in dynamic balance, it is called homeostasis. When this is disturbed the cell is under stress. Kind of stress: Biological stress: viruses, bacteria, fungi, parasites Chemical stress: cell poisons e.g. toxins, acids, heavy metals Physical stress: temperature, radiation, pressure The effect of the stress might be: Cytocidic - cell will be killed Cytostatic - cell will survive, but its cell cycle will be halted Mitoclastic - mitosis will be stopped Mutagenic or genotoxic - DNA will be damaged Stress reaction - mobilization of the cell (changes in gene expression) that will lead to the production of specific proteins characteristic of the exact type of the stress, e.g. the high temperature will change the protein conformation (the structure), the cell produces HSPs (heat shock proteins), that help to repair changed conformation. 14.1. Physical stress TEMPERATURE Every cell or organism has its own temperature optimum range (biokinetic temperature). Organisms preferring very high temperature are referred to as thermophilic, compared to cryophylic or psychrophilic organisms that prefer low temperatures. When the temperature is outside the optimum temperature range, the organism (cell) is under temperature stress. If temperature is increased above the optimum (heat shock), the secondary, tertiary and quaternary protein structure can be destroyed, leading e.g. to defect of biomembranes, the failure of enzymes or the damage of the mitotic spindle. If temperature is decreased under the optimum, the membranes become rigid and their permeability decreases; mitotic spindle (microtubules) is damaged. The low temperature is used for cryoconservation (of sperm, tissues etc.) and lyophilization (or cryodesiccation), when the sample is not only frozen but also dehydrated. RADIATION 1) VISIBLE LIGHT usually does not do any harm to the organism (cell). Unfortunately, the organism can be sensitized toward the day light by chemical compounds. This effect is called photodynamy and chemical compounds causing sensitization to light are called photodynamic colors e.g. hypericin in Hypericium perforatum, phagopyrin in buckwheat, eosin, fluorescein, tar (also used in shampoos) or porphyrin (metabolite of haemoglobin that can cause disease called cutaneous porphyria). In humans, cutaneous porphyria is disease caused by deficiency in the enzymes (the main precursors of heme pathway) that leads to insufficient production of heme and accumulation of porphyrins (toxic to tissue in high concentrations). The porphyria affects the skin causing photosensitivity (photodermatitis), blisters, necrosis of the skin and gums, swelling. In some forms of porphyria, heme precursors are excreted in the urine, causing changed coloration. Heme precursors may also accumulate in the teeth and fingernails, giving them a reddish appearance. 2) IONIZING RADIATION - its effect depends on the type of ionizing radiation, its intensity, the impact time, and sensibility of the cell (organism). Mitotic cells (e.g. in bone marrow, lymphatic tissue, epithelium, or germinal cells) are highly sensible to radiation, 92 while non-dividing cells (e.g. neural or muscle cells blocked in G0 phase) are quite resistant. 3) UV RADIATION causes both indirect DNA damage (free radicals and reactive oxygen species generation, that contributes e.g. to skin cancer development) and damage of the DNA bases (mutagenic effect). The radiation excites DNA molecules, leading preferentially to aberrant covalent bonds formation between adjacent cytosine bases (pyrimidine dimers). The presence of pyrimidine dimers inhibits DNA replication. The UV radiation is also used in laboratories for sterilisation (in germicide lamps). ___________________________________________________________________________ TASKS TASK 1: Photodynamy NP: hay infusion, eosin Prepare two slides, one with one drop of hay infusion, the other with two drops. Add eosin to one of the two drops on one slide (experiment) and to the single drop on the other slide (control). Expose the slide with two drops to light and place the other slide into dark. Observe specimen after 3 min. intervals and compare it to controls. The photodynamy causes excitation of the ciliates and their rapid movement, followed by inhibition and death. (hay infusion + eosin) (hay infusion) In the dark (hay infusion + eosin) In the light TASK 2: The effect of ionizing irradiation on the testes of rat and fowl PP: testes of rat (normal tissue and tissue after irradiation with dose of 6.5 Gy) “varle krysa, varle krysa 6,5 Gy”, testes of fowl (normal tissue, tissue 4 and 8 days after irradiation with doses of 12 Gy) - “varle kohout, varle kohout - 4 dny, varle kohout - 8 dnů”. Specimens are stained with haematoxylin-eosin. Observe the testes under small and then larger magnification and compare the normal tissue with the tissue after irradiation. You can find damaged tubules and space around them. It is difficult to differentiate individual stages of spermatogenesis inside the tubules, because cells are damaged. In fowl, you can observe signs of reparation eight days after irradiation. 10 4 A B Seminiferous tubule Fig. 72: The effect of ionizing irradiation on the testes of rat: A – normal tissue (small and large magnification), B – tissue after irradiation (small and large magnification). 93 14.2. Chemical stress Substances which cause chemical stress are also called toxins or xenobiotics. They cause toxic shock to a cell (organism). The effect depends on the kind and the structure of the chemical substance, on its concentration, on the impact time and on the sensibility of the cell (organism). Xenobiotics can be divided according to various criteria: According to the chemical composition: Heavy metals Acids, bases Akaloids According to their origin: Biological – snake venom, bee poison (apisin), botulotoxin, aflatoxin (from fungi), etc. Synthetic – DDT, pesticides, etc. According to the mechanism of action: Attack at the synthesis of biopolymeres that causes defects in the DNA replication and mutations can occur (some antibiotics - actinomycin D, toxin from mushrooms of the genus Amanita). Changes in the transport through membrane which can increase permeability of the biomembrane and leads to the lysis of cell (peptides from bee and snake venoms) or to increased influx of potassium into the cell and thus changed ratio of the ions inside the cell (some antibiotics, e.g. valinomycin). Detergents damage the structure of biomembrane less specifically. Attack at the energetic metabolism by blocking some enzymes of anaerobic glycolysis (cyanides), blocking oxidative phosphorylation (toxins that damage the structure of biomembranes) or by direct binding of toxin to ATP molecule. Attack of the cell cycle by blocking S-phase, mitosis and/or cytokinesis (colchicine that binds to the microtubules thus disabling their polymerisation and blocking the mitotic spindle. PHYTONCIDES are antimicrobial organic compounds derived from various plant spices e.g. onion, garlic, horseradish, oak and pine trees. Phytoncides (allicin and alliin in onion, allicin and diallyl disulfide in garlic) prevent plants from rotting, defend the plant surrounding from bacteria, fungi and insects and have also effect on ciliates. OLIGODYNAMIC ACTION is effect of small amounts of chemical substances (e.g. metallic ions such as cadmium and mercury) in the medium to inhibit the growth of bacteria (or other microorganisms), to inactivate enzymes or even to kill the microorganisms. ___________________________________________________________________________ TASKS TASK 1: Oligodynamic action (the effect of heavy metal ions dissolved in water solution (water upon mercury) on a survival of Protozoa NP: hay infusion, water upon mercury (Hg) Put a drop of hay infusion and a drop of water upon the mercury on the slide. Observe a specimen in 3 min. interval and compare it with control (only hay infusion). You can observe excitation of ciliates and their rapid movement, followed by inhibition and death. Control (hay infusion) Experiment (hay infusion + water upon mercury) 94 TASK 2: The effect of CdCl2 (cadmium chloride) on the testes of rat PP: testes of rat (normal tissue and tissue of rat that was fed with CdCl2) - “varle po CdCl2”. Specimens are stained with haematoxylin-eosin. Observe the testes of a rat that was fed with CdCl2 (under small and large magnification) and compare it with normal testes. Focus on the seminiferous tubules (their number and structure) and spermatogenesis. TASK 3: The effect of sublimate (HgCl2 - bichloride of mercury or perchloride of mercury) on survival of Tubifex tubifex Use Petri dishes and prepare four different dilutions of sublimate in water according to the table and one control (only water). Then place about five Tubifex tubifex to each dish, observe their reactions and note times of their death. dish no. water (in ml) 0.15 % HgCl2 (in ml) 1 0 10 2 3 7 3 5 5 4 7 3 5 10 0 TASK 4: The effect of detergent on a survival of Tubifex tubifex Use Petri dishes and prepare four different dilutions of detergent with water and control (only water). Place about five Tubifex tubifex to each dish, observe reactions of the heddles and note times of their death. dish no. water (in ml) detergent (in ml) 1 0 10 2 3 7 3 5 5 4 7 3 5 10 0 TASK 5: The effect of phytoncides on survival of Protozoa NP: hay infusion Cut an onion into small pieces and then crush it using sea sand and a mortar. Drop hay infusion on the slide and observe the presence of protozoa. Then put the specimen into the Petri dish and place crushed onion under it (see the picture). Control reactions and movement of the protozoa in 3-5 min. intervals and compare it with control (hay infusion without the effect of phytoncides). Note the time of protozoa’s death. Hay infusion Crushed onion Control Hay infusion 95 15. Genetics Genetics is biological discipline that studies heredity and variation in living organisms. The understanding of the process of inheritance began, among others, with the work of Johann Gregor Mendel in the mid-nineteenth century. Since that times, the physical basis for heredity (genes) have been explored. Genes correspond to regions within DNA, the sequence of which carries the genetic information organisms inherit. The sequence of nucleotides in a gene is translated (to produce protein) according to a set of rules called genetic code. Genetic code is the key that enables translation of information encoded in genetic material into proteins by living cells. The genetic code defines the relation between tri-nucleotide sequences of DNA (and subsequently mRNA) called codons, and amino acid sequence. Every triplet of nucleotides in a gene coding sequence specifies a single amino acid and is “red” by an anticodon, a triplet on tRNA molecule. There are 64 possible codons (four possible nucleotides at each of three positions = 4 3) and only 20 standard amino acids, hence the genetic code is redundant and multiple codons can specify the same amino acid. The correspondence between codons and amino acids is nearly universal among all known living organisms. The direction of the flow of genetic information between different macromolecules is described by the so-called central dogma of molecular biology that was first enunciated by Francis Crick in 1958. In general, genetic information is transferred between DNA molecules by their copying (DNA replication) and it can be transferred into mRNA by transcription, and into proteins by translation on ribosomes using the information in mRNA as a template for protein synthesis, according to genetic code. Reverse transcription (the transfer of information from RNA to DNA) occurs e.g. in retroviruses, such as HIV. Information cannot be transferred back from protein to nucleic acid. The DNA of all organisms contains both genes and non-coding sequences, such as various regulatory or repetitive sequences. In many species, only a small fraction of the total sequence of the genome encodes protein (e.g. only about 1.5 % of the human genome consists of proteincoding exons), while other organisms, such as prokaryotes have much higher proportion of coding DNA. Genes encode either for proteins or for tRNA or rRNA molecules. Most eukaryotic genes (unlike in prokaryotes) are composed of non-coding DNA sequences called introns and of coding regions called exons. Introns are transcribed to precursor mRNA (premRNA), but later are removed by a process called splicing, thus they are absent in mature mRNA and are not translated into proteins. Regulatory elements, such as promoters, termintors, enhancers or silencers regulate gene expression. Changes to the nucleotide sequence are generally referred to as mutations, that can be spontaneous (caused e.g. by errors during replication) or induced (e.g. by exposure to UV or ionizing radiation, chemical mutagens, or viruses). In multicellular organisms, mutations can be subdivided into germ line mutations, which can be passed on to descendants, and somatic mutations (mutations in somatic cells). According to the extent of the change (or the effect on DNA structure), the following types of mutations are recognised: 1) Small-scale mutations affecting one or a few nucleotides include: Point mutations, that exchange a single nucleotide for another: transitions that exchanges a purine for a purine (A ↔ G) or a pyrimidine for a pyrimidine (C ↔ T) or transversions, which exchange a purine for a pyrimidine and vice versa (C/T ↔ A/G). Point mutations that occur within the protein coding region of a gene may be classified into three kinds (depending on the type of change in codon): silent mutations result in a codone which codes for the same amino acid; missense mutations code for a different amino acid and nonsense mutations produce stop codon that terminates translation, thus producing truncated protein. 96 Insertions add one or more extra nucleotides into the DNA. Insertions in the coding region of a gene may cause a shift in the reading frame (frameshift), which can significantly alter the gene product. Insertions can be reverted by excision of the transposable element. Deletions remove one or more nucleotides from the DNA and thus can also alter the reading frame of the gene. 2) Large-scale mutations affecting chromosomal structure: Amplifications lead to multiple copies of a chromosomal region. Deletions of larger chromosomal segments can lead to genetic disorders, such as Cri du chat syndrome caused by truncated short arm of chromosome 5. Translocations are caused by interchange of genetic material from nonhomologous chromosomes. Inversions reverse the orientation of a chromosomal segment. 3) Genome mutations: changes in the number of chromosomes (aneuploidies and euploidies) 15.1. Cytogenetics - study of chromosomes, karyotypes CHROMOSOMES are organised structures of DNA and proteins that are found in eukaryotic cells. Their size is about 1-10 μm. DNA contains many genes, regulatory elements and other nucleotide sequences. In eukaryotes, DNA-bound proteins (histones) serve to package the DNA into a material called chromatin and to control its functions. The structure of chromosomes and chromatin varies through the cell cycle. Chromosomes may exist as either unduplicated as single linear strands (in G1 phase of cell cycle) or duplicated as two identical DNA copies (chromatides) joined by a centromere. Chromosomes are duplicated by replication during the S phase of the cell cycle. Based on the position of centromere, the chromosomes can be classified as telocentric, acrocentric, submetacentric or metacentric. The structure of chromatin varies significantly between different stages of the cell cycle, according to the requirements of the DNA. Chromosome shorter arms are called p, while the longer ones are called q arms. At the end of eukaryotic chromosomes, there are regions of repetitive DNA called telomeres, which protect the ends of the chromosome. Nukleosom (DNA with histones) DNA Fig. 73: Location of DNA in the nucleus of the cell. 97 The word chromosome comes from the Greek (chroma = color and soma = body) due to their property of being stained very strongly by some dyes during mitosis. During the interphase, two types of chromatin can be distinguished according to intensity of DNA condensation, staining and transcription: decondensed euchromatin (light-colored, active transcription) and more tightly condensed heterochromatin (stained darkly, consists of mostly inactive DNA). Heterochromatin can be further distinguished into two types: constitutive heterochromatin (never expressed DNA sequences located mainly around the centromere that usually contains repetitive sequences) and facultative heterochromatin (which is sometimes expressed). During mitosis, the whole chromosomes are highly condensed. Sexually reproducing species have somatic cells (body cells), which are diploid (2n) having two sets of chromosomes (one from the mother and one from the father). Gametes (reproductive cells) are haploid (n) with one set of chromosomes. Somatic cells are produced by mitosis, gametes are produced by meiosis. There are two types of chromosomes: autosomes (somatic chromosomes, homologous chromosomes) and gonosomes (sex chromosomes, heterologous chromosomes). For example, human somatic cells are diploid and have 46 chromosomes in total, 22 different pairs of autosomes and two gonosomes (the same or different – see below). Gametes are haploid and have 23 chromosomes in total, 22 autosomes and one gonosome. The total number and structure of chromosomes is species-specific. Some plant species are polyploid: they have more than two sets of homologous chromosomes. SEX DETERMINATION SYSTEMS An organism's sex is usually defined by the gametes it produces: males produce male gametes while females produce female gametes. Individuals (species) with differentiated sexes are referred to as gonochorists, while organisms which produce both male and female gametes are termed hermaphroditic (e.g. snails and the majority of flowering plants). The biological cause for an organism developing into one sex or the other is called sex determination. Sexes can be determined either by genetic or non-genetic factors (e.g. environmental temperature). In genetic sex determination systems, different sexes can be determined by type and number of sex chromosomes (gonosomes) or by number of sets of somatic chromosomes (autosomes). Genetic sex determination, because it is determined by chromosome assortment, usually results in approximately 1:1 ratio of male and female offspring. 1) Genetic sex determination systems: Type XY (Mammal, Drosophila) (sex chromosomes X and Y) ZW (Bird, Abraxas) (sex chromosomes Z and W) XX/XO (Protenor) (sex chromosome X) Haplodiploidy (number of chromosomes sets) Male Female Representative mammals, insect, some fish, reptiles and amphibians XY XX ZZ ZW birds, butterflies, some fish, reptiles, plants, amphibians XO XX true bugs and orthopteran insects n 2n social insects 2) Non-genetic sex determination systems: Temperature-dependent sex determination: In reptiles which do not have sex chromosomes (alligators and some turtle species), the gender of offspring is influenced by temperature of egg incubation (temperature <28 °C result only in males, temperature 28-32 °C results in males and females and temperature >32 °C results only in females). 98 Behaviour sex determination: In some species of marine fish (e.g. clown fish), sex can be changed during the life of an individual. Sex change is initiated by removal or addition of dominant male or female to the population. No sex determination: Some species have no sex determination, either because they are hermaphrodites, or because all the individuals are females and reproduce by parthenogenesis (some species of lizards, fish or insects). BARR BODY, LYONIZATION. In those species (including humans) in which sex is determined by two different sex chromosome (XY or ZW), a Barr body (sex chromatin) is the inactive X (or Z) chromosome in a female or male somatic cells, respectively. Barr body, named after the discoverer Murray Barr, is a condensed and after staining densely colored oval body (1 μm) in the periphery of the nucleus. The process of one sex chromosome inactivation during embryogenesis in homogametic sex is called lyonization. The choice of which X chromosome will be inactivated is random, but once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell and remains condensed even in interphase, when other chromosomes are decondensed. There is the possibility of reactivation of inactive X chromosome during gametogenesis. In humans, the Barr body is visible in 20-70 % cells in females. POLYTENE (GIANT) CHROMOSOMES. Some specialized cells undergo repeated rounds of DNA replication without cell division (endomitosis), forming a giant polytene chromosomes (chromatids remain synapsed together). Polytene chromosomes are 50-200× longer then normal chromosomes; their length is 200-600 μm and width 25 μm. They have characteristic light and dark banding patterns (chromomeres) because of different staining of heterochromatin and euchromatin. Chromosome puffs are diffuse uncoiled regions of the polytene chromosome, where transcription takes place. Polytene chromosomes were originally observed in the larval salivary glands of Chironomus midges by Balbiani in 1881. The hereditary nature of these structures was studied in Drosophila melanogaster. They are known to occur in the secretory tissues of other dipteran insects such as Malpighian tubules of Sciara and also in protists, plants, mammals, or in cells from other insects. CYTOGENETICS is a branch of genetics that is concerned with the study of chromosomes and cell division. It includes routine analysis of G-banded chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Cells from bone marrow, blood, amniotic fluid, cord blood, tumour, and tissues (including skin, umbilical cord, liver, and many others) can be cultured using standard cell culture techniques in order to increase their number. Mitotic activity of the cells is stimulated by fytohemaglutinin (bean extracted). After cultivation for 72 hours in 37 oC, a mitotic inhibitor (colchicine, colcemid) is added to the culture. This stops the cell division at mitosis, yielding to increased number of mitotic cells for analysis. The cells are placed into a hypotonic solution, which causes the cells to swell so that the chromosomes will spread when placed on a slide. Subsexuently the cells are fixed and subjected to banding and analysis. KARYOGRAM, IDIOGRAM is a diagram of all chromosomes of one cell arranged in pairs and ordered by size and position of centromere. KARYOTYPE is the characteristic chromosome complement of a eukaryotic species or individual. The normal human karyotypes contain 22 pairs of autosomes and one pair of sex chromosomes. Normal karyotypes for women contain two X chromosomes and are denoted 46,XX; men have both an X and a Y chromosome denoted 46,XY. Variations 99 from the standard karyotype usually lead to developmental abnormalities. Karyotypes are usually used to study chromosomal aberrations, such as aneuploidies. EUPLOIDY is the change in number of chromosome sets e.g. triploidy (3n), hexaploidy (6n). ANEUPLOIDY is the change in number of individual chromosomes, e.g. monosomy (2n-1), trisomy (2n+1). Aneuploidies occur as a result of nondisjunction during meiosis in the formation of a gamete. Extra or missing chromosomes are among the most widely recognized genetic disorders in humans, causing various syndromes with specific symptoms. The most frequent and well-known syndroms in humans are: Down syndrome – trisomy of 21 chromosome, in both genders (47XX+21; 47XY+21) Edwards syndrome – trisomy of 18 chromosome, in both genders (47XX+18; 47XY+18) Patau syndrome – trisomy of 13 chromosome, in both genders (47XX+13; 47XY+13) Turner syndrome – monosomy of X chromosome, only in females (45, X0; or 45, X) Klinefelter syndrome – extra X chromosome in males (47, XXY) Syndrome of three X (metafemale) – extra X chromosome in females (47, XXX) XYY syndrome (Jacobs syndrome, metamale) – extra Y chromosome in males (47, XYY) CHROMOSOME-BANDING TECHNIQUES o G-banding (Giemsa banding) uses trypsin (to partially digest the proteins) followed by Giemsa staining. This creates unique banding patterns on the chromosomes with dark (adenine and thymine rich, gene poor) and light bands. o Q-banding (Quinacrine banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope. o R-banding (Reverse banding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. o FISH (fluorescence in situ hybridization) - refers to using fluorescently labelled DNA probes to hybridize (based of complementarity of the sequences) to cytogenetic cell preparations. Analysis of FISH specimens is done by fluorescence microscopy. It is possible to use multicolor FISH (M-FISH) with more differently colored DNA probes to detect more DNA sequences or Spectral karyotyping (SKY) technique to simultaneously visualize all the pairs of chromosomes in an organism in different colors. ___________________________________________________________________________ TASKS TASK 1: Sex chromatin in somatic cells NP and PP: buccal smear - “pohlavní chromatin” Take a sample from the inner side of the cheek using a cotton swab and impress the swab on a slide. Fix it above the flame and apply acetorcein on the slide for 5 min. and then cover with a cover glass. In somatic cells of female buccal mucosa, you can find sex chromatin (Barr body), a densely colored body in the periphery of the nucleus. Nucleolus Nucleolus 10 40 Sex chromatin (Barr body) Fig. 74: Sex chromatin. 100 TASK 2: Structure of polytene (giant) chromosome PP: polytene (giant) chromosome - “obrovské chromosomy”, NP: salivary gland of chironomid larva (Chironomus sp.) or Drosophila melanogaster Prepare salivary glands of the chironomid larva and control the presence of the giant chromosomes under a microscope. Pass the slide (sample-side up) through the flame of the burner 3 to 4 times to heat-fix the sample. Stain it with acetorcein for 5-10 min. Cover with a cover glass and prepare the compression sample. Observe polytene chromosomes with chromomeres (stripes of different width) and puffs. Larva of chironomid Salivary gland Polytene chromosome Chromomere s Fig. 75: Polytene chromosome in cells of salivary gland in chironomid larva. TASK 3: Mammal karyotype PP: karyotype of rabbit, pig, sheep, horse, cattle and human - “karyotyp králík, prase, ovce, kůň, skot, člověk”. Observe permanent samples prepared from lymphocytes of different domestic animals or human (Note: In the samples, you can find round nucleus of lymphocytes and clusters of chromosomes = karyotype). Compare the numbers and types of chromosomes (rabbit 2n = 44, pig 2n = 38, sheep 2n = 54, horse 2n = 64 and human 2n = 46). TASK 4: Karyotype of onion NP: the rootlet of onion (Allium cepa) cultivated with 0.02 % water solution of colchicin and stained with acetorcein Prepare the compression sample from a stained onion rootlet and find the cluster of chromosomes (karyotype). Chromosome 10 s 40 A B Fig. 76: Examples of karyotype: A – in cattle (Bos taurus), B – in onion (Allium cepa). 101 TASK 5: Karyotype records Write following karyotypes: a) b) c) d) karyotype of healthy man and woman karyotype of bull (male cattle), mare (female horse), ram (male sheep) karyotype of man with Down syndrome and man with Klinefelter syndrome karyotype of woman with Edwards syndrome and woman with Turner syndrome 15.2. Model organism - Drosophila melanogaster Drosophila melanogaster is a small fly, belonging to the Diptera and often called "fruit fly" (or vinegar fly, wine fly, etc.). The genus, however, contains about 1,500 species and is very diverse in appearance, behaviour and breeding habitat. Drosophila is found all around the world, with more species in the tropical regions. They can be found in deserts, tropical rainforest, cities, swamps, and alpine zones. Some northern species hibernate. Most species breed in various kinds of decaying plant and fungal material, including fruit, bark, slime fluxes, flowers, and mushrooms. A few species have switched to being parasites or predators. Several Drosophila species, including D. melanogaster, D. immigrans, and D. simulans, are closely associated with humans, and are often referred to as domestic species. Drosophila melanogaster has been heavily used in research in genetics and is a common model organism in developmental biology. Drosophila melanogaster is a popular experimental animal because it is small and easy to grow in the laboratory, has a short generation time with many offspring, it has a small genome (about 13 600 genes) with only four pairs of chromosomes: three autosomes, and one sex chromosome (karyotype 2n = 8), and mutant animals are readily obtainable. In 1906 Thomas Hunt Morgan began his work on D. melanogaster and reported his first finding of a white eyed mutant in 1910. His work on Drosophila earned him the 1933 Nobel Prize in Medicine for identifying chromosomes as the vector of inheritance for genes. Life cycle: development from fertilisation till adult individual lasts approximately from 7 to 19 days, depending on the temperature. Fruit flies undergo complete metamorphosis. Egg (one day) - white, size 0.5 mm 1st instar larva (one day) 2nd instar larva (one day) 3rd instar larva (two days) Pupa (five days) - white, later brown Adult (imago) (40-50 days) with size 2-3 mm, male is smaller with dark spots at the rounded abdomen, female is bigger with dark stripes at the sharpened abdomen; unfertilized female (4-6 hours after hatching) is lighter with elongated abdomen and sometimes with not quite developed wings. The female becomes fertile about ten hours after hatching, copulates with male and lays about 300 eggs 6-10 days after copulation. Laboratory cultures: fruit flies are cultured in Erlenmeyer flasks under room temperature or in the thermostat with 25 oC. At the bottom of the flask, there is medium prepared from maize grit, dry yeast, sugar and agar, covered with filtrate paper. The flask is closed with cotton plug. Mutant strains: there are several mutations in different genes encoding for different traits (e.g. eye color, body color, morphology of wings) in fruit fly. Recessive mutant allele is marked with small letter (w „white“= white eyed; y „yellow“= yellow body), dominant mutant allele is marked with capital letter (B „bar“= narrowed eye), standard allele is marked with symbol (+ = e.g. red eyed). 102 Mutation white vestigial curly yellow ebony eyeless Wild form forma Symbol w vg cy y e ey White Phenotype white-colored eyes vestigial wings curly wings yellow-colored body black-colored body reduced eyes Vestigial Curly Yellow Ebony Fig. 77: Wild form and some types of mutations in fruit fly (Drosophila melanogaster). ___________________________________________________________________________ TASKS TASK 1: Genetic experiment Genetic experiment - crossing of red-eyed drosophila (original wild form) with mutant wite-eyed drosophila. Experiment will proceed during three lessons: The beginning of experiment (1st lesson) - You will be divided into groups. Each group will receive an Erlenmeyer flask with a fresh medium (maze grit, dry yeast, sugar and agar) and two tubes with drosophila of different sexes (male, female) and different eye color (red, white). Observe them and then carefully place females and males with different eye colors together into the Erlenmeyer flask. Mark the flask with the date, sex and eye colors of crossed drosophila and identification of your group. Removing of adult drosophila (2nd lesson) - Drop ether on the cotton placed at the cover of the bottle and close it. Wait a few minutes and subsequently remove the adult fruit flies from the Erlenmeyer flask into the bottle with ether to narcotize them. Evaluation of experiment (3rd lesson) - Use ether to narcotize fruit flies (offspring of crossing) and sort them according to sex and eye color; deduce how the observed trait (eye color) is inherited. TASK 2: Mutant strains Observe different mutant strains of drosophila (white, yellow, ebony, curly and vestigial) - live, in alcohol or on the picture. 15.3. Monohybridism GENE is a unit of inheritance, encodes for one protein (structural gene) or for tRNA or rRNA molecule. ALLELE is a concrete form of gene (can be dominant e.g. A or recessive a). LOCUS (plural loci) is a fixed position of gene on chromosome. GENOME is set of DNA in nucleus (mitochondrial genome is set of DNA in mitochondria; chloroplast genome is set of DNA in chloroplast). 103 GENOTYPE is a set of all genes in organism or the genetic (allelic) constitution of organism with respect to trait. TRAIT (character) is a feature of an organism (e.g. blood groups, color of hair). PHENOTYPE in common use it is synonym for trait, strictly speaking it indicates the state of trait (e.g., for trait eye color there are phenotypes: blue, brown and green) or it is a complex of traits in organism produced by genotype (in conjunction with environment). HOMOZYGOTE is an individual with two same alleles of certain gene (it can be dominant AA or recessive aa) HETEROZYGOTE (hybrid) is an individual with two different alleles of certain gene (Aa). P GENERATION (parental) is a generation of parents, that are different homozygotes (dominant and recessive). F1 GENERATION (first filial) is the first generation of uniform offspring (hybrids, heterozygotes), the result of crossing of two parents from P generation. F2 GENERATION (second filial) is the second generation of offspring, the result of crossing of two individuals from F1 generation. B1 GENERATION (back crossing) is the first generation of crossing of homozygote from P generation (usually recessive homozygote) with heterozygote from F1 generation. COMPLETE DOMINANCE is relation between alleles, when heterozygote has the same phenotype as dominant homozygous individual. INCOMPLETE DOMINANCE (semidominance) is relation between alleles, when heterozygote has different (intermediate) phenotype compared to both dominant homozygous and recessive homozygous individuals. MONOHYBRIDISM (MONOHYBRID CROSS) is crossing with one studied trait. JOHANN GREGOR MENDEL (1822 – 1884) was an Augustinian priest and scientist, who is often called “the father of genetics”. He lived in Moravia, which was part of the Austrian Habsburg Monarchy in his times. From his hybridization experiments especially with garden peas (Pisum sativum) he deduced two generalizations, that later became known as Mendel's Laws of Heredity. He published his findings in 1865 and 1866, but his work was not understood and was almost forgotten. It was "re-discovered" in 1900 by Hugo de Vries, Carl Correns and Erich von Tschermak, who formulated the so-called Mendel’s principles. MENDEL’S PRINCIPLES (MENDEL’S LAWS) 1. Mendel’s First Law of Heredity (or principle of segregation) states that (1) alternative alleles of a gene are discrete (do not blend in heterozygote); (2) when gametes are formed by heterozygous diploid individual, the two alleles segregate from one another; and that (3) each gamete has an equal probability of possessing either member of an allele pair. This law is also referred to as principle of uniformity of F1 hybrids and principle of identity of reciprocal crosses. 2. Mendel’s Second Law of Heredity (or principle of independent assortment) states that genes located on different chromosomes assort independently of one another during meiosis). Mendel´s principles hold true in the following condition: 1) monogenic inheritance (one gene encodes for one trait) 2) autosomal inheritance (genes are located on autosomes) 3) genes are located on different chromosome pairs CHÍ SQUARE TEST ( 2 test) is a statistic method used for determination of significance of variation between obtained (empiric) and expected (theoretic) values. In genetics, it is used for comparison of obtained and expected genotypic (phenotypic) ratios of monitored trait. 104 2 (N) ei ) 2 (x i ei xi….obtained (empiric) value ei …expected (theoretic) value N….degree of freedom (number of cleavage classes - 1) The calculated value is compared with the table value (supplement no.1) for the relevant degree of freedom N (number of cleavage classes - 1) at desired significance level P. If calculated value for 2 is smaller than the table value, then variation between obtained and expected values (genotypic ratios) is not statistically significant, it means that the trait was cleaved in ratio that we have expected. The most commonly used significance level (“power”) is P ≤ 0.05; meaning that the result will be statistically significant with 95 % probability. ___________________________________________________________________________ TASKS TASK 1: In snapdragon plants (Antirrhinum maius), dominant homozygote has red flowers, recessive homozygote has white flowers and heterozygote has pink flowers. a) Complete genotypes in P (parental), F1 (first filial) and F2 (second filial) generations. What is the phenotype and genotype ratio of F1 and F2 generations? b) What type of inheritance does the observed trait show? (complete or incomplete dominance) TASK 2: Color of pumpkin can be white or yellow. Dominant allele W determines white color, while recessive allele w determines yellow color. a) After crossing of two white pumpkins, 3/4 of offspring were white and 1/4 of offspring were yellow. What are the genotypes of parents and offspring? b) White phenotype of pumpkin can be encoded by two different genotypes. Which are they? How can you find the genotype of one particular white pumpkin? TASK 3: Two black female mice were crossed with two brown males. One female had 9 black and 7 brown offspring in several litters; the second female had 57 black offspring in several litters. a) How is the coat color inherited in mice? What color is dominant and why? Explain. b) What are the genotypes of parents and offspring from the aforementioned crossings? TASK 4: Leaves of cucumber can be palmate or flabellate (called ginkgo). After crossing of two plants (homozygote with palmate leaves and homozygote with flabellate leaves), all offspring were palmate. a) What type of leaves is dominant (palmate or flabellate)? a) What are genotype and phenotype ratios in F2 and B1 (back crossing = crossing of recessive homozygote and heterozygote) generations? TASK 5: In Andalusian strain of chicken, dominant allele B determines dark color of feather, while recessive allele b determines white color of feather. Heterozygote has bluish color of feather. What will be the color of feather of offspring after crossing of bluish female with: a) dark colored fowl, b) bluish colored fowl, c) white fowl? 105 TASK 6: Use 2 test to compare obtained phenotype ratio in F2 generation (79 red, 170 pink and 95 white snapdragon plants) with expected ratio (see task 1). Table values are in supplement no. 1. TASK 7: White pumpkin crossed with yellow one resulted in 327 white and 361 yellow offspring (see task 2). a) What are the genotypes of parents and offspring? b) How is this type of crossing called? c) Compare obtained and expected ratios using χ2 test (table value in supplement no.1). TASK 8: Blue and brown color of eyes in humans is determined by two different alleles of one gene. In 337 families, the following data were found: Parents (color of eyes) blue x blue blue x brown brown x brown Families 150 158 29 Children (color of eyes) blue brown 625 0 317 322 25 82 What color of eyes is dominant? Complete genotypes of parents and children. 15.4. Dihybridism, polyhybridism and branching method DIHYBRIDISM is the crossing with two studied traits. POLYHYBRIDISM is the crossing with three or more studied traits. PUNNETT SQUARE is a diagram designed by Reginald Punnett and used by biologists to determine the probability of offspring having particular genotype and phenotype. It is made by establishing all possible combinations of alleles coming from female and male gametes. F1 generation: AaBb × AaBb Gametes: AB:Ab:aB:ab AB:Ab:aB:ab F2 generation: Gamets AB Ab aB ab AB AABB AABb AaBB AaBb Ab aB ab AABb AaBB AaBb AAbb AaBb Aabb AaBb aaBB aaBb Aabb aaBb aabb There are 16 possible combinations of alleles after the crossing of two dihybrids in F2 generation. There are rules how to write Punnett squares and genotype/phenotype ratios. You should start with the left upper corner and continue to the right lower corner. Then genotypes in F2 generation are: AABB: AABb: AAbb: AaBB: AaBb: Aabb: aaBB: aaBb: aabb. Genotype ratio is: 1: 2: 1: 2: 4: 2: 1: 2: 1. Phenotype ratio in case of complete dominance is: 9A-B- : 3 A-bb: 3 aaB- : 1 aabb (dominant or recessive alleles can be completed instead of dash). In Punnett square, there is one diagonal with hybrids (AaBb) and the other diagonal with homozygotes (AABB, AAbb, aaBB, aabb). Two central genotypes (AAbb, aaBB) are new combinations of alleles and express new phenotypes that were not present in previous generation. BRANCHING METHOD is method used for setting genotypes and genotypes/phenotype ratios without using Punnett square. It is especially suitable in case of polyhybridism. The principle is in the gradual branching of different alleles or pairs of alleles of all genes. 106 Example 1: Use the branching method for setting of genotypes of gametes produced by individual with the following genotype: AaBBCc. Genotypes of gametes: AB C ABC c ABc aB C aBC c aBc Individual with this genotype can produce four different gametes. Example 2: Use the branching method for setting of genotype and genotype ratios in offspring of parents with these genotypes: Aabbcc × AaBbCC (there is the relation of complete dominance between the two alleles of all genes). Aa x Aa …AA, 2Aa, aa bb x Bb…..Bb, bb cc x CC…..Cc Genotypes of offspring: AA BbCc AABbCc bbCc AAbbCc 2Aa BbCc 2AaBbCc bbCc 2AabbCc aa BbCc aaBbCc bbCc aabbCc Parents with those genotypes can produce offspring with six different genotypes in genotype ratio: 1: 1: 2: 2: 1: 1. ___________________________________________________________________________ TASKS TASK 1: Complete Punnett square for dihybridism and deduce phenotype ratio for offspring of two guinea-pigs with the same genotype RrBb (R - rough coat, r smooth coat, B - black coat, b - white coat). There is the relation of complete dominance between the two alleles of both genes. What offspring results from the back crossing? TASK 2: Use the branching method for setting of genotypes of gametes produced by individuals with the following genotypes: a) RrssTtUU b) AaBBCcddEe c) KkllmmNnOOppQq TASK 3: Use the branching method for setting of genotype and phenotype ratios in offspring of parents with these genotypes: RrssTtUU x RrSsTTuu (between alleles of all genes is complete dominance). TASK 4: Use the method of branching for setting of genotype and phenotype ratio in offspring of parents with these genotypes: AaBBCcddEe x aaBBCcDdee (There is the relation of incomplete dominance between the two alleles of all genes). TASK 5: In pumpkins, white color (W) is dominant over yellow (w) and disk shape (D) is dominant over round shape (d). What are the genotypes of parents and offspring in the following crossings? a) White disk x yellow round (offspring: 1/2 white disk and 1/2 white round). 107 b) White disk x yellow round (offspring: 1/4 white disk, 1/4 white round, 1/4 yellow disk and 1/4 yellow round). c) White disk x white disk (offspring: 28 white disk, 9 white round, 10 yellow disk and 3 yellow round). TASK 6: Black rough and white rough guinea-pigs had 32 black rough, 33 white rough, 12 black smooth and 9 white smooth offspring (see task 1). a) What were genotypes of both parents? b) Use the branching method for determination of genotype frequencies of offspring. c) Use 2 test for determination of significance of variation between obtained (empiric) and expected (theoretic) phenotype ratios (table value - supplement no. 1). TASK 7: In hen, feathery legs (F) are dominant over featherless legs (f) and pea comb (P) is dominant over simple comb (p). Two cocks (A and B) were mated with two hens (C a D). All of them had feathery legs and pea combs. Cock A mated with hens C and D had all offspring with feathery legs and pea combs. Cock B mated with hen C had offspring with feathery or featherless legs and only pea combs, while mated with hen D offspring had only feathery legs and pea or simple combs. What are the genotypes of parents (cocks and hens) and their offspring? 15.5. Polymorphic genes POLYMORPHIC GENE (from Greek “polys” = many, “morphe” = a form or shape) exists in two or more allelic forms. It has been estimated that 20 to 50 % of all structural gene loci in humans occur in more allelic forms, e.g. alleles coding for different eye colors or different ABO blood groups in humans. CODOMINANT ALLELES - two different alleles of one gene are responsible for different phenotypes, both affecting phenotype of heterozygote. E.g. main blood group system (so-called AB0) is determined by one gene (I). In human population there are three alleles (IA, IB, i) and four phenotypes (A, B, AB, 0 blood types). Allele A and B are co-dominant, both contribute to phenotype of heterozygote. ___________________________________________________________________________ TASKS TASK 1: In rabbits, there is allele set with dominance in this order: colored fur C, Himalayan albinism ch and albinism ca. a) What color of fur can be expected in offspring after crossing of two homozygotes one with colored fur and the second with Himalayan albinism? b) What are the genotypes of rabbits with colored fur and Himalayan albinism, if they had these offspring: 1/2 colored, ¼ Himalayan albinism and ¼ albinism? TASK 2: In humans, blood groups are determined by three alleles IA, IB and i. Alleles I and I are dominant over allele i. There is relation of codominance between IA and IB (presence of these two alleles results in blood group AB). What blood groups are expected in offspring of parents with genotypes I Ai and IBi? A B 108 TASK 3: What are the genotypes of parents, if father had blood group AB, mother B and their children ¼ A, ¼ AB and ½ B? TASK 4: Which of two men can be excluded as a father of child with blood group 0? Mother of the child had blood group B, one man had blood group A and second man had blood group AB. 15.6. Gene interactions GENE INTERACTION is the participation of two or more different genes in production of one phenotypic character. Monogenic inheritance, condition of Mendelian principles, is not fulfilled. The cleavage ratio is changed in F2 generation. RECIPROCAL INTERACTION - the trait is present in more forms; each of them is encoded by one combination of parent alleles of concerned genes. This gene interaction is without change of cleavage ratio. DOMINANT EPISTASIS - dominant allele of one gene (epistatic) suppresses expression of dominant allele of second gene (hypostatic). RECESSIVE EPISTASIS - recessive homozygous constitution of one gene (epistatic) suppresses expression of dominant allele of second gene. INHIBITION - dominant allele of inhibitor gene suppresses the manifestation of dominant allele of other gene. Inhibitor gene itself has no effect on the phenotype. Change of cleavage ratios in F2 generation in different gene interactions: Gene interactions Reciprocal interaction Dominant epistasis Recessive epistasis Complementarity Compensation Inhibition Duplicity noncumulative Duplicity cumulative with dominance (complete dominance) A-B- A-bb aaB- aabb 9 3 3 1 12 3 1 9 3 4 9 7 10 3 3 13 3 15 1 9 6 1 AaBb AaBB aaBB Aabb AABB AABb AAbb aaBb aabb Duplicity cumulative without dominance (incomplete dominance) 1 4 6 4 1 COMPLEMENTARITY - dominant alleles of two (or more) genes cooperate in realization of phenotype. The this trait is expressed only if at least one dominant allele of both genes is present at the same time. COMPENSATION - function of dominant alleles of two different genes is contradictory, their phenotype effects exclude each other. DUPLICITY (or MULTIPLICITY in case of more than two genes) is interaction of two or more genes with the same effect on phenotype. The intensity of effect depends on if the effects of genes cumulate or not and if there is relationship of dominance between alleles of particular gene. The genes are very often minor genes (polygenes). 109 LETHAL GENES are genes whose expression results in the death of the organism, usually during embryogenesis. ___________________________________________________________________________ TASKS TASK 1: Purple color of vetch (wild pea) is caused by the presence of dominant alleles of both genes (C-P-). Others combination of genotypes encodes for white color of flowers. a) What is the phenotype ratio in F2 generation? Use Punnett square. What type of gene interaction is it? b) What color of flower can be expected in offspring resulting from the following crossings: 1) CcPp x CcPP 2) Ccpp x ccPp? TASK 2: In pumpkin, the shape is determined by two genes: A and B. The pumpkin is round if dominant allele of gene A or of gene B is present in the genotype, it is discoid if dominant alleles of both genes are present, and it is elongated in individuals who are recessive homozygotes in both genes. a) What is the phenotype ratio in F2 generation (crossing of two dihybrids)? Use combination square. What type of gene interaction is it? b) The crossing of the discoid pumpkin with the round pumpkin resulted in 3/8 discoid, 1/2 round and 1/8 elongated offspring. What were the genotypes of parents and of offspring? TASK 3: The color of a feather of a canary is determined by genes A and B. Dominant allele of gene A encodes for red color, dominant allele of gene B for yellow one. Birds with genotypes aabb and A-B- are white. a) What is the phenotype ratio in F2 generation? What type of gene interaction is it? b) Third gene (C) determines, if the feather is smooth or fuzzed. Birds with dominant allele C are smooth, recessive genotype determines fuzzed feather. Use branching method to find phenotype ratio in offspring resulting from crossing: AaBbCC x AabbCc. TASK 4: In a pumpkin, the orange color is determined by a dominant allele W, white color by dominant allele Y. Plants with genotypes W-Y- and W-yy are orange, wwY- white and wwyy green. a) What is the phenotype ratio in F2 generation? Use Punnett square. What type of gene interaction is it? b) What will be the color and phenotype ratio of the offspring after crossing of parents with genotypes: WwYy and Wwyy? TASK 5: The color of a feather of a budgerigar (Melopsittacus undulatus) is determined by the interaction of two genes: F and O. Allele F encodes for yellow color (genotype F-oo) and allele O encodes for blue color (genotype ffO-). If both alleles are present, budgerigar is green (genotype F-O-). Recessive homozygote in both genes is white (genotype ffoo). a) What is the phenotype ratio in F2 generation (crossing of two dihybrids)? Use combination square. What type of gene interaction is it? b) What will be the feather color and phenotype ratios in the offspring after crossing of parents with genotypes: 1) FFOo x ffOo 2) FfOO x Ffoo? 110 c) The crossing of yellow and blue budgerigars resulted in 6 yellow and 5 green offspring. What are the genotypes of parents and offspring? d) Green female budgerigar had white offspring. What was the genotype of female and offspring? TASK 6: In mice, the presence of dominant allele of gene C is important for production of dark pigment melanin. Dominant allele of gene A causes change of dark pigment into yellow. a) What is the phenotype ratio in F2 generation? Use Punnett square. What type of gene interaction is it? b) What offspring will result from crossing of black mouse (CCaa) with white mouse (ccAA)? TASK 7: In chicken, dominant allele of gene A determines colored feather, dominant allele of gene I suppresses effect of gene A, but has no effect on the phenotype. a) What is the phenotype ratio in F2 generation (crossing of two dihybrids)? Use Punnett square. What type of gene interaction is it? b) What feather can be expected in offspring resulting from crossing: AaIi x Aaii? TASK 8: Scaliness in carp (Cyprinus carpio) is determined by genes S and N. Between these genes, there is reciprocal interaction. The following phenotypes can exist: row (S-Nn), scaly (S-nn), smooth (ssNn), bare (ssnn). The presence of two dominant alleles N in the genotype is lethal. a) What are the phenotypes in offspring resulting from crossing of two row carps (SsNn x SsNn)? b) Use the branching method to find phenotype ratio in offspring resulting from crossing of row and smooth carps. TASK 9: In humans, the color of hair is determined by the interaction of six genes. Gene A encodes for pigment formation and is recessive epistatic against other genes (it means that man with genotype aa is albino). Gene B encodes for formation of brown pigment and is dominant epistatic against R gene (it means that man with genotype bb has fair hair). Gene R encodes for formation of red pigment (recessive allele r is inactive). Dominant alleles D, F, V influence intensity of hair color. In all six genes, there is complete dominance between alleles of individual genes. Use the branching method to find phenotypes of offspring, if their parents had black hair and genotypes: AaBBRrDDFfVv and AaBBRrDDFfVv TASK 10: The coat color of some rodents is determined by the interactions of three genes: A, B and C. Dominant allele of gene C causes albinism and is recessive epistatic against genes A and B. Between genes A and B, there is reciprocal interaction. Dominant allele A determines gray color, recessive allele determines black color. Dominant allele B determines yellow pigmentation of hair ends ("wild color"); recessive allele b has no effect on the phenotype. Use the branching method to find phenotypes of offspring, if their parents had genotypes: AabbCc x AaBbcc. 111 15.7. Inheritance and sex SEX-LINKED INHERITANCE – the genes are located on gonosomes (sex chromosomes), thus autosomal inheritance (the condition of Mendel’ principles) is not valid. Sex-linked traits can show both dominant and recessive inheritance pattern. Most of the sex-linked traits (including diseases such as haemophilia or Duchenne muscular dystrophy) are recessive i.e. they are expressed in the absence of dominant allele. Sex-linked dominant traits are rarer. They should be considered more deleterious because most are male lethal. An example of an X-linked dominant trait in cattle is Streaked Hairlessness in Holsteins. This disorder causes streaks of missing hair, especially on the flanks. Males, which inherit this allele, die in utero. Complete sex-linked traits - genes are located on heterologous part of sex chromosomes. a) The gene is located on heterologous part of Y chromosome - holandric inheritance (trait is inherited from father to son), e.g. hypertrichosis auriculae (excessive hair growth on ear auricles). b) The gene is located on heterologous part of X chromosome - trait exists in both sexes, e.g. haemophilia or daltonisms in man, color of eyes in fruit fly. Individuals that have only one copy of X- or Y-linked gene (males in mammals) are referred to as hemizygous. X-linked genes show the following features: Uniformity of F1 generation, if dominant allele is on X chromosome in female (XDXD) in P generation. Cross inheritance, if dominant allele is on X chromosome in male (XDY) in P generation. Incomplete sex-linked traits - genes are located on homologous part of sex chromosomes (recombination is possible, but crossing-over is usually blocked). XX Homologous part of chromosomes Heterologous part of chromosomes Fig. 78: Simplified diagram of sex chromosomes X and Y with marked homologous and heterologous parts. SEX-LIMITED INHERITANCE – the genes are located on autosomes (somatic chromosomes) of both sexes, but the trait is expressed only in one sex that has an anatomic predisposition (e.g. antlers in deer, or cryptorchism in humans = absence of one or both testes in scrotum). SEX-INFLUENCED INHERITANCE – the genes are located on autosomes of both sexes; phenotype in heterozygote is influenced by sex of carrier due to the presence of male or female sex hormones (e.g. male pattern baldness). Sex-influenced traits appear to be “dominant” in males and “recessive” in females. SEX-CONTROLLED INHERITANCE – the genes are located on autosomes of both sex, phenotype is controlled by sex hormones in heterozygote and dominant homozygote (e.g. secondary sex traits - beard in man). __________________________________________________________________________________________ TASKS TASK 1: What are phenotype ratios in F2 generation of fruit flies when a red-eyed female is crossed with a white-eyed male and on other side, a white-eyed female is crossed with a red-eyed male? 112 TASK 2: In cats, dominant allele of gene Y (in homozygote form in females and hemizygote form in males) determines black color of fur. Recessive allele determines yellow color and heterozygote females have tortoise-colored fur. a) What type of inheritance is it? On which chromosome is gene for fur color located? b) Black female has one tortoise-colored and four black offspring. What genotype and color of fur does their father have? What sex and color does the black offspring have? c) Tortoise-colored female was mated with yellow male. What is probability that they will have yellow male and female offspring? TASK 3: Daltonism (color blindness) is disease with recessive X-linked inheritance. A couple has daughter with daltonism, but her mother is able to distinguish colors. What genotypes do parents and their daughter have? TASK 4: Haemophilia is disease with a recessive gonosomal inheritance linked to chromosome X. A man with haemophilia has daughter with healthy woman with homozygote genotype. What genotype does their daughter have? TASK 5: The color of Ayrshire cattle is determined by gene M. Cattle and bull a) b) c) d) with genotype MM are mahogany, cattle with recessive homozygote genotype is red spotted. The bull with genotype Mm is mahogany, while the cattle is red spotted. What type of inheritance is it? The red spotted cattle (her father was mahogany) was mated with the red spotted bull. What are genotypes and phenotypes of parents and their offspring? What is the phenotype ratio in F2 generation? The mahogany cattle gave birth to red spotted offspring. What is the sex of offspring? TASK 6: In humans, there is gene P for baldness (hairlessness). Men with genotype PP and Pp are bald, while women are bald only with genotype PP. Gene B determines color of eyes; brown one is dominant over blue one. a) What type of inheritance does hairlessness show? b) The hairless man with brown eyes (his father has hair and blue eyes) married a blond woman with blue eyes (her father and brothers were bald). What color of eyes can they expect in their children? Will they have hair or will they be bald? TASK 7: In Siamese fighting fish (Betta splendens), dominant allele of gene Z induces enlargement of fins, if there are male sex hormones presents. a) What type of inheritance is it? b) What type of fins can we expect in offspring resulted from crossing: Zz x Zz. c) How can you identify genotype of male with enlarged fins and genotype of female, if you have recessive homozygotes for crossing? 113 15.8. Genetic linkage GENETIC LINKAGE occurs when particular genes (or alleles) are located on the same chromosome. Mendel´s principle of independent assortment does not hold true in this case. Genes located on one chromosome are physically connected to form linkage group and segregate together during meiosis (if they are not separated by crossing over). Genetic linkage was first discovered by the British geneticists William Bateson and Reginald Punnett shortly after Mendel's laws were rediscovered, and was later studied also by Thomas Hunt Morgan, who was the first person awarded the Nobel Prize thanks to discoveries on the field of genetics (findings concerning the role played by the chromosome in heredity). Complete linkage - small distance between genes, strong linkage and small probability of crossing-over. Incomplete linkage - large distance between genes, weak linkage and large probability of crossing-over. MORGAN´S PRINCIPLES (LAWS): 1. Genes are located on one chromosome in linear order 2. Number of linkage groups equals to a number of pairs of homologous chromosomes POWER OF LINKAGE is a probability of crossing-over between alleles of different genes that are in linkage. The greater the distance between linked genes is, the weaker linkage and the greater the chance that non-sister chromatids would cross over in the region between the genes. The power of linkage is characterised by the following numbers: BATESON NUMBER (c) is determined as a proportion of a number of offspring with nonrecombinant (parental) genotype (or phenotype) and number of offspring with recombinant (different from parents) genotype (or phenotype). c number of offspring with non - recombinan t phenotype number of offspring with recombinan t phenotype MORGAN NUMBER (p) is determined as a percentage of offspring with recombinant genotype (or phenotype) from all offspring. Centimorgan (cM) is a unit of recombinant frequency for measuring genetic linkage and the distance between genes. One centimorgan is defined as the genetic distance between two loci with a statistically corrected recombination frequency of 1 % (1 cM corresponds to a physical distance of about one million base pairs, which is roughly about 0.003 mm). Centimorgan is now more commonly called “map unit” (mu) or locus map unit (LMU). p 100 x number of offspring with recombinan t phenotype (cM) number of all offspring c 100 - p p p 100 c 1 TEST CROSSING (three-point cross) is similar to back crossing (crossing of heterozygote and homozygote). Phenotype ratio in offspring is used to find out the order of genes and their distances. Three-point cross is used to determine relations between three genes. 114 Example 1: What is the order of genes R, S and T and power of linkage (distance) between neighbouring genes based on genetic analysis of offspring resulted from the test crossing: RrSsTt × rrsstt. Procedure: Phenotype Frequency 1) Set the correct order of genes based on phenotype with (%) the lowest frequency (this phenotype is the result RST 78.5 of double crossing over). In the table, there is the lowest rst frequency in the last phenotype, but if you draw it, there RsT 14.4 is not double crossing (see Fig.). It means that RST is not rSt correct order. You have to change the order of genes on Rst 6.7 both chromosomes to obtain double crossing over. rST Correct order of genes is RTS. rsT 0.4 2) Correct order of genes in all phenotypes (see Fig.) RSt 3) Calculate the linkage power (p) for genes RT and TS. The linkage power is connected with frequency of crossing over. When we look at the picture after correction, there is crossing over between RT in two last phenotypes, so p(RT) = 6.7 + 0.4 = 7.1 cM. For genes TS, there is crossing over between genes in the second and last phenotype, so p(TS) = 14.4 + 0.4 = 14.8 cM. 4) Draw a chromosome map with the correct order of genes and their distances. . R S T R T S r s t r t R s T R T s r S t r t S R s t R t s r S T r T S T No double crossing-over t r T s Double crossing-over t S r s R S After correction R s Chromosome map R T 7.1 cM S 14.8 cM ___________________________________________________________________________ TASKS TASK 1: What genotypes can we expect in gametes, if there is incomplete linkage of genes A and B in gametogony with genotype AaBb? TASK 2: What is the phenotype ratio in B1 generation resulting from crossing: AB × ab ab ab Genetic linkage p(AB) = 16.6 cM and number of offspring n = 1152. 115 TASK 3: In hens, feathery legs are dominant over featherless (gene A), pea comb over simple (gene B) and white color over dark (gene C). What is the order of genes ABC and power of linkage between neighbouring genes based on genetic analysis of offspring (n = 2500) resulted from test crossing: AaBbCc x aabbcc. Draw chromosome map. Frequency in Phenotypes offspring (%) Test crossing: ABC ABC × abc 80.9 abc abc abc ABc 3.9 abC Abc 14.6 aBC AbC 0.6 aBc TASK 4: What is the order of the four genes K, L, N and S and power of linkage (distance) between neighbouring genes located on 5. chromosome of tomato? (K-red color, k-yellow; L-rounded shape, l-sharp; N-pilous stalk, n-bare; S-straight leaf, s-curly). Phenotype frequencies (%) of offspring resulted from test crossing are in tables. Use three-point cross and draw chromosome map of all four genes. Phenotype NKS nks NkS nKs Nks nKS nkS NKs % Phenotype KSL ksl kSl KsL KSl ksL Ksl kSL 67.8 29.2 2.2 0.8 % 49.6 21.4 20.4 8.6 15.9. Population genetics POPULATION is a group of organisms of one species that can interbreed, live at the same place and at the same time, and that have the common ancestor. GAMETE POOL is set of gametes in population. GENE POOL (GENOFOND) is set of genes (alleles) in population. ALLELIC FREQUENCY is frequency of certain allele in population. GENOTYPE FREQUENCY is frequency of certain genotype in population. PANMICTIC POPULATION is a population with individuals that interbreed without limitation; there is random mating. HARDY-WEINBERG EQUILIBRIUM (HW law) - indicates relation between allelic and genotypic frequencies in population. In enough large panmictic population, there are no allelic and genotypic frequency changes from one to the other generation, if there is no selection, mutation, genetic drift, and migration (genetic flow). For allele frequency: p(A) q(a ) 1 (p is frequency of dominant allele, q is frequency of recessive allele) 116 For genotype frequency: P(AA) H(Aa) Q(aa) 1 (P is frequency of dominant homozygote, H is frequency of heterozygote, and Q is frequency of recessive homozygote) Hardy-Weinberg equilibrium For gene with two alleles: (p + q)2 = p2 + 2pq + q2 = 1 allele frequencies genotype frequencies For gene with three alleles: (p + q + r)2 = p2 + 2pq + q2 + 2pr + 2qr + r2 = 1 Allelic frequencies can be derived from genotype frequency and vice versa. __________________________________________________________________________ TASKS TASK 1: Gene I (AB0 blood system) exists in three forms - IA, IB and i. What are the genotype and phenotype frequencies in panmictic population, if frequency of allele IB (q) = 0.4 and frequency of allele i (r) = 0.4? TASK 2: In humans, three types of earlobes can be found. In a group of 100 students, free earlobe (hanging free from the head) was found in 38 students (genotype dominant homozygotes), middle attached in 45 students (heterozygotes) and attached (joined to the head) in 17 students (recessive homozygotes). a) What are the frequencies of dominant and recessive alleles? b) Use χ2 test to verify, if population is in HW equilibrium. TASK 3: Population is in HW equilibrium. Frequency of allele (b) for blue color of eyes is 0.6. What is the frequency of blue-eyed people in the population? How many % of brown-eyed people with homozygote and heterozygote genotypes are in this population? TASK 4: In humans, the presence of RhD antigen (named after rhesus monkey), determines the blood type. If the antiserum against this antigen agglutinates your red blood cells you are Rh+ (Rh positive) (genotype DD or Dd), if it doesn't you are Rh (Rh negative) (genotype dd). In the Central European Caucasian population, approximately 84 % of people are Rh+ and 16 % Rh-. What is the frequency of the dominant allele? TASK 5: Frequency of recessive allele (a) for myopy (short sightedness) in a population is q (a) = 0.14. What are the frequencies of short-sighted people (genotype aa) and of healthy carriers (genotype Aa) in this population? TASK 7: Albinism is recessively inherited disorder (inability to synthesize pigment melanin). In the population, there is one albino (aa) in 10 000 people (the frequency of genotype aa is 0.0001). a) What is the allelic frequency of recessive allele a? b) How many % of carriers (Aa) are present in this population? 117 15.10. Quantitative genetics QUANTITATIVE TRAIT is a characteristic that varies in degree and can be attributed to the interactions between two or more genes (polygenic inheritance) and their environment. An example of quantitative trait in humans is height, skin color or body mass. POLYGENIC INHERITANCE, also known as quantitative or multifactorial inheritance refers to inheritance of a phenotypic characteristic (trait) that is attributable to two or more genes and their interaction with the environment. Polygenic traits do not follow patterns of Mendelian inheritance. Instead, their phenotypes typically vary along a continuous gradient depicted by a bell curve. Many diseases, such as autism, cancer or diabetes show polygenic inheritance pattern with contribution of environmental factors. HERITABILITY estimates the relative contributions of genetic and non-genetic factors to the total phenotypic variance in a population. Heritability is determined by broad-sense heritability coefficient or narrow-sense heritability coefficient. Values of heritability coefficients run from 0 (phenotypic variance is caused only by environmental factors) to 1 (or 100 %) (phenotypic variance is caused only by genetic variance). There can be traits with low heritability values (<0.2), e.g. body weight of poultry, middle heritability values (from 0.2 to 0.5), such as body length in pigs and traits with high heritability values (>0.5), e.g. butterfat percentage in cattle. Broad-sense heritability coefficient h2B - reflects all possible genetic contributions to a population's phenotypic variance. (VG is genetic variance due to allelic variation = additive variance, due to dominance variation, gene interactions, as well as maternal and paternal effects; VP is phenotypic variance) 2 Narrow-sense heritability coefficient h N (VA is additive genetic variance, VP is phenotypic variance) h 2B VG VP h 2N VA VP The basic statistic characteristics used for study of inheritance of quantitative traits: ARITHMETIC AVERAGE x (arithmetic mean) 2 VARIANCE S and STANDARD DEVIATION S determine range of values distributed around average. xi x xi s2 n x 2 s2 s n 1 CORRELATION AND CORRELATION COEFFICIENT r determines if there is any relation between monitored quantitative traits. Values of correlation coefficient can range from -1 to +1. The closer to one, the stronger correlation between traits is. Plus values mean positive correlation (continual proportion between traits); while minus values mean negative correlation (reciprocal proportion between traits). Following formulas are used to calculate correlation coefficient. cov xy xi x yi n 1 y xi yi 1 xi n n 1 118 yi r cov xy s xs y REGRESSION COEFFICIENT k is used to calculate narrow-sense heritability h2N. Values of regression coefficient can be from 0 to 1 (0 = full additive effect, 1 = no additive effect). k cov xy s x2 __________________________________________________________________________ TASKS TASK 1: Evaluate the variability of weight in two groups of mice and compare the results. Use the arithmetic average and variance. First group: 15.5 g, 10.3 g, 11.7 g, 17.9 g, 14.1 g Second group: 20.2 g, 21.2 g, 20.4 g, 22.0 g, 19.7 g TASK 2: In eight ducks, width of head and length of wing was measured (table). Duck 1 2 3 4 5 6 7 8 Width of head (cm) 2.75 3.20 2.86 3.24 3.16 3.32 2.52 4.16 Length of wing (cm) 30.3 36.2 31.4 35.7 33.4 34.8 27.2 52.7 a) Count the arithmetic average and standard deviation for these two traits (width of head and length of wing). b) Use the correlation coefficient to find out what correlation is between the two traits. TASK 3: The height of parents (average of father and mother) and their children was measured (table). Height of children (cm) 175 180 177 160 165 175 185 175 183 Height of parents (cm) 175 190 180 175 175 173 195 185 172 a) Count the arithmetic average and the variance for the height of children and parents. b) Use the correlation coefficient to find out what correlation is between the height of parents and children. c) Count the narrow-sense heritability for the height (use regression coefficient). TASK 4: Heritability of height in wheat h2N = 0.7. There is a trend to reduce the height of wheat by selection. Will this goal be reached promptly or slowly? TASK 5: The height in humans has heritability h2N = 0.9. How much is the phenotype influenced by the environmental conditions? 119 16. Recommended literature Alberts B. et al.: Essential Cell Biology, Garland Science, Taylor and Francis Group, 2004. Campbell N. A., Reece J. B.: Biology, Pearson, 2004. Page R. D. M., Holmes E.: Molecular evolution. A phylogenetic approach. Blackwell Science Ltd., Oxford, 1998. Ringo J.: Fundamental Genetics. Cambridge University Press, 2004. Gregory T.: The Evolution of the Genome, Academic Press, 2004, Elsevier Inc. http://www.sciencedirect.com/science/book/9780123014634 Syvanen M. and Kado C. I.: Horizontal Gene Transfer (Second Edition), 2002, Elsevier Ltd. http://www.sciencedirect.com/science/book/9780126801262 Hickman C. P., Jr., Roberts L. S., Larson A., I'Anson H.: Integrated principles of zoology. McGraw-Hill Education, 2003. 120 Supplement no 1: Values of chi square test ( 2) for significance levels P = 0.95 - 0.001 and for degrees of freedom N = 1 - 30. N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 0.95 0.004 0.103 0.35 0.71 1.15 1.63 2.17 2.73 3.32 3.94 4.57 5.23 5.89 6.57 7.26 7.96 8.67 9.39 10.12 10.85 11.59 12.34 13.09 13.85 14.61 15.38 16.15 16.93 17.71 18.49 0.90 0.016 0.21 0.58 1.06 1.61 2.2 2.83 3.49 4.17 4.87 5.58 6.30 7.04 7.79 8.55 9.31 10.09 10.87 11.65 12.44 13.24 14.04 14.85 15.66 16.47 17.29 18.11 18.94 19.77 20.6 0.80 0.064 0.45 1.01 1.65 2.34 3.07 3.82 4.59 5.38 6.18 6.99 7.81 8.63 9.47 10.31 11.15 12.00 12.86 13.72 14.58 15.45 16.31 17.19 18.06 18.94 19.82 20.70 21.59 22.47 23.36 0.70 0.15 0.71 1.42 2.20 3.00 3.83 4.67 5.53 6.39 7.27 8.15 9.03 9.93 10.82 11.72 12.62 13.53 14.44 15.35 16.27 17.18 18.10 19.02 19.94 20.87 21.79 22.72 23.65 24.58 25.51 0.50 0,.6 1.39 2.37 3.36 4.35 5.35 6.35 7.34 8.34 9.34 10.34 11.34 12.34 13.34 14.34 15.34 16.34 17.34 18.34 19.34 20.34 21.34 22.34 23.34 24.34 25.34 26.34 27.34 28.34 29.34 0.30 1.07 2.41 3.67 4.88 6.06 7.23 8.38 9.52 10.66 11.78 12.90 14.01 15.12 16.22 17.32 18.42 19.51 20.60 21.69 22.78 23.86 24.94 26.02 27.10 28.17 29.25 30.32 31.39 32.46 33.53 121 0.10 2.71 4.61 6.25 7.78 9.24 10.65 12.02 13.36 14.68 15.99 17.28 18.55 19.81 21.06 22.31 23.54 24.77 25.99 27.20 28.41 29.62 30.81 32.01 33.20 34.38 35.56 36.74 37.92 39.09 40.26 0.05 3.84 5.99 7.82 9.49 11.07 12.59 14.07 15.51 16.92 18.31 19.68 21.03 22.36 23.69 25.00 26.30 27.59 28.87 30.14 31.41 32.67 33.92 35.17 36.42 37.65 38.89 40.11 41.34 42.56 43.77 0.02 5.41 7.82 9.84 11.67 13.39 15.03 16.62 18.17 19.68 21.16 22.62 24.05 25.47 26.87 28.26 29.63 31.00 32.35 33.69 35.02 36.34 37.66 38.97 40.27 41.57 42.86 44.14 45.42 46.69 47.96 0.01 6.64 9.21 11.34 13.28 15.09 16.81 18.48 20.09 21.67 23.21 24.73 26.22 27.69 29.14 30.58 32.00 33.41 34.81 36.19 37.57 38.93 40.29 41.64 42.98 44.31 45.64 46.96 48.28 49.59 50.89 0.001 10.83 13.82 16.27 18.47 20.52 22.46 24.32 26.13 27.88 29.59 31.26 32.91 34.53 36.12 37.70 39.25 40.79 42.31 43.82 45.32 46.80 48.27 49.75 51.18 52.6 54.05 55.50 56.89 57.45 59.70